Anti-EphA4 Antibody

ABSTRACT

Provided is an anti-EphA4 antibody that can bind to EphA4 and enhance the cleavage of EphA4, as well as a pharmaceutical composition comprising the antibody as the active ingredient. 
     A mouse anti-EphA4 antibody that has binding affinity towards EphA4 and can enhance the cleavage of EphA4 was obtained, and the sequence of the complementarity-determining region (CDR) of the mouse anti-EphA4 antibody was identified. Subsequently, the anti-EphA4 antibody of interest was obtained by producing a humanized antibody of the mouse anti-EphA4 antibody.

TECHNICAL FIELD

The present invention relates to an antibody that binds to EphA4, anucleic acid that encodes the antibody, a vector that comprises thenucleic acid, a cell comprising the vector, a method of producing theantibody, and a pharmaceutical composition comprising the antibody.

BACKGROUND ART

EphA4 is a member of the receptor tyrosine kinase family. Ephrin type Aand type B are known as ligands for EphA4, and when EphA4 binds withephrin which is a ligand thereof, de-adhesion signal is induced. So far,the involvement of EphA4 in the pathology of Alzheimer's disease(hereinafter also referred to as “AD”) has been suggested (Non-PatentLiteratures 1-4), and it has been reported that the inhibition ofbinding between EphA4 and ephrin rescues amyloid β (Aβ)₍₁₋₄₂₎olygomer-mediated dysfunction of neurotransmission (Patent Literature1). In AD, it is thought that aggregates (neurofibrillary tangles)formed by excessively phosphorylated tau are involved in nerve celldeath (Non-Patent Literature 5), and it has also been reported thatsuppression of tau phosphorylation suppresses neurodegeneration ofsynapse disappearance (Non-Patent Literature 6 and Non-Patent Literature7), as well as improves memory deficits or cognitive dysfunction(Non-Patent Literatures 8-11). There are reports suggesting thatactivation of CDK5 is a cause of tau phosphorylation (Non-PatentLiterature 12 and Non-Patent Literature 13). A genetically modifiedmouse expressing a P301L mutation which has been found in familialfrontotemporal dementia (rTg4510 mouse) is an AD model mouse, andsimilarly to AD, hyperphosphorylation of tau and abnormal accumulationof tau in neuronal cells are found in the mouse. In rTg4510 mouse,neurofibrillary tangles, a pathological feature of AD, are formed andbring cognitive dysfunction by brain atrophy and loss of neuron(Non-Patent Literature 14 and Non-Patent Literature 5).

EphA4 is abundantly expressed in the hippocampus or the cerebral cortex,and is neural activity-dependently cleaved by matrix metalloprotease(MMP), ADAM (a disintegrin and metalloproteinase), and γ selectase. Itis known that this cleavage reaction of EphA4 stabilizes the spine whichis a key structure in neural function (Non-Patent Literature 15). It hasbeen reported that the density of spine is decreased in AD (Non-PatentLiterature 16), and since decrease of cleaved fragments of EphA4 is alsoconfirmed in AD at NFT stages V and VI, it is thought that EphA4cleavage reaction is involved in the pathology of AD (Non-PatentLiterature 17).

Although KYL peptide and compound 1 etc. are known as existing EphA4inhibitory drugs (Patent Literature 2, Non-Patent Literature 18, andNon-Patent Literature 19), there has been no reports regarding anantibody having activity that enhances the cleavage of EphA4.

CITATION LIST

-   [Patent Literature 1] WO2016/019280A1-   [Patent Literature 2] WO2012/156351A1-   [Non-Patent Literature 1] Vargas L M et al., PLoS One. 2014 Mar. 21;    9 (3)-   [Non-Patent Literature 2] Fu A K et al., Proc Natl Acad Sci USA.    2014 Jul. 8; 111 (27): 9959-64-   [Non-Patent Literature 3] Rosenberger A F et al., Acta Neuropathol    Commun. 2014 Jul. 16; 2: 79-   [Non-Patent Literature 4] Huang T Y et al., J Exp Med. 2017 Dec. 4;    214 (12): 3669-3685.-   [Non-Patent Literature 5] Santa Cruz et al., Science. 2005 Jul. 15;    309 (5733): 476-81-   [Non-Patent Literature 6] Seo J et al., J Neurosci. 2017 Oct. 11; 37    (41): 9917-9924-   [Non-Patent Literature 7] Patrick G N et al., Nature. 1999 Dec. 9;    402 (6762): 615-22.-   [Non-Patent Literature 8] Onishi T et al., J Neurochem. 2011    December; 119 (6): 1330-40-   [Non-Patent Literature 9] Belfiore R et al., Aging Cell. 2019    February; 18 (1): e12873.-   [Non-Patent Literature 10] Webster S J et al., Front Genet. 2014    Apr. 23; 5: 88.-   [Non-Patent Literature 11] Grayson B et al., Behav Brain Res. 2015    May 15; 285: 176-93.-   [Non-Patent Literature 12] Cancino G I et al., Neurobiol Aging. 2011    July; 32 (7): 1249-61.-   [Non-Patent Literature 13] Vargas L M et al., Biochim Biophys Acta    Mol Basis Dis. 2018 April; 1864: 1148-1159.-   [Non-Patent Literature 14] Ramsden M et al., J Neurosci. 2005 Nov.    16; 25 (46): 10637-47.-   [Non-Patent Literature 15] Inoue E et al., J Cell Biol. 2009 May 4;    185 (3): 551-64-   [Non-Patent Literature 16] Boros et al., Ann Neurol. 2017 October;    82 (4): 602-614-   [Non-Patent Literature 17] Matsui C et al., Brain Pathol. 2012    November; 22 (6): 776-87. doi: 10.1111/j.1750-3639-   [Non-Patent Literature 18] Goldshmit et al., PLoS one. 2011; 6 (9):    e24636-   [Non-Patent Literature 19] Van Hoecke et al., Nature Medicine. 2012    September; 18 (9): 1418-22, 2012

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The object of the present disclosure is to provide an anti-EphA4antibody that can bind to EphA4 and enhance the cleavage of EphA4, aswell as a pharmaceutical composition comprising the antibody as theactive ingredient.

Means for Solving the Problems

As a result of extensive investigation to solve the above problems, thepresent inventors obtained a mouse anti-EphA4 monoclonal antibody thatcan bind to EphA4 and enhance the cleavage of EphA4, and produced ahumanized antibody of the antibody to thereby come to complete theantibody of interest.

The present disclosure encompasses the following characteristics.

(1) An anti-EphA4 antibody, wherein

the anti-EphA4 antibody comprises heavy and light chains, and comprises:

(a) a heavy chain CDR1 consisting of the amino acid sequence shown inSEQ ID NO. 44;(b) a heavy chain CDR2 consisting of the amino acid sequence shown inSEQ ID NO. 27;(c) a heavy chain CDR3 consisting of the amino acid sequence shown inSEQ ID NO. 28;(d) a light chain CDR1 consisting of the amino acid sequence shown inSEQ ID NO. 29;(e) a light chain CDR2 consisting of the amino acid sequence shown inSEQ ID NO. 30; and(f) a light chain CDR3 consisting of the amino acid sequence shown inSEQ ID NO. 31.

(2) The anti-EphA4 antibody according to (1), wherein the anti-EphA4antibody is humanized.

(3) The anti-EphA4 antibody according to (1) or (2), wherein theanti-EphA4 antibody specifically binds to EphA4 and enhances thecleavage of EphA4.

(4) The anti-EphA4 antibody according to any of (1)-(3), wherein theanti-EphA4 antibody specifically binds to EphA4 and inhibits the bindingbetween EphA4 and ephrin.

(5) The anti-EphA4 antibody according to any of (1)-(4), wherein

the variable region of the heavy chain consists of the amino acidsequence shown in SEQ ID NO. 45, and

the variable region of the light chain consists of the amino acidsequence shown in SEQ ID NO. 46.

(6) The anti-EphA4 antibody according to any of (1)-(5), wherein

the constant region of the heavy chain and the constant region of thelight chain comprise amino acid sequences derived from a human antibody.

(7) The anti-EphA4 antibody according to (6), wherein

the constant region of the heavy chain is the constant region of humanIgG.

(8) The anti-EphA4 antibody according to (7), wherein the constantregion of human IgG is the constant region of human IgG₂.

(9) The anti-EphA4 antibody according to (8), wherein

the constant region of human IgG₂ comprises the amino acid sequenceshown in SEQ ID NO. 47.

(10) The anti-EphA4 antibody according to any of (6)-(9), wherein

the constant region of the light chain is the constant region of humanIgκ.

(11) The anti-EphA4 antibody according to (10), wherein

the constant region of human Igκ comprises the amino acid sequence shownin SEQ ID NO. 48.

(12) An anti-EphA4 antibody, wherein

the anti-EphA4 antibody comprises heavy and light chains,

the heavy chain comprises the amino acid sequence shown in SEQ ID NO.59, and

the light chain comprises the amino acid sequence shown in SEQ ID NO.60.

(13) The anti-EphA4 antibody according to (12), wherein

the C-terminal lysine of the heavy chain is deleted.

(14) An anti-EphA4 antibody, wherein

the anti-EphA4 antibody comprises heavy and light chains,

the heavy chain comprises the amino acid sequence shown in SEQ ID NO.59,

the light chain comprises the amino acid sequence shown in SEQ ID NO.60, and

the C-terminal lysine of the heavy chain is deleted.

(15) An isolated nucleic acid encoding the anti-EphA4 antibody accordingto any of (1)-(14).

(16) A vector comprising the nucleic acid according to (15).

(17) A host cell comprising the vector according to (16).

(18) A method of producing an anti-EphA4 antibody, comprising a step ofculturing the host cell according to (17).

(19) A pharmaceutical composition comprising the anti-EphA4 antibodyaccording to any of (1)-(14).

(20) A pharmaceutical composition according to (19), wherein thepharmaceutical composition comprises at least one pharmaceuticallyacceptable carrier.

(21) The pharmaceutical composition according to (19) or (20), fortreating Alzheimer's disease.

(22) The anti-EphA4 antibody according to any of (1)-(14) for use in thetreatment of Alzheimer's disease.

(23) A method for treating Alzheimer's disease, comprising administeringto a patient in need thereof a therapeutically effective amount of theanti-EphA4 antibody according to any of (1)-(14).

(24) Use of the anti-EphA4 antibody according to any of (1)-(14) formanufacturing a pharmaceutical composition for treating Alzheimer'sdisease.

(25) The pharmaceutical composition according to (19) or (20), fortreating tauopathy.

(26) The anti-EphA4 antibody according to any of (1)-(14) for use in thetreatment of tauopathy.

(27) A method for treating tauopathy, comprising administering to apatient in need thereof a therapeutically effective amount of theanti-EphA4 antibody according to any of (1)-(14).

(28) Use of the anti-EphA4 antibody according to any of (1)-(14) formanufacturing a pharmaceutical composition for treating tauopathy.

(29) The pharmaceutical composition, the anti-EphA4 antibody, the methodfor treating, or the use according to any of (25)-(28), wherein thetauopathy is Alzheimer's disease or frontotemporal lobar degenerationwith tau pathology.

(30) The pharmaceutical composition, the anti-EphA4 antibody, the methodfor treating, or the use according to (29), wherein the tauopathy isAlzheimer's disease.

(31) The pharmaceutical composition, the anti-EphA4 antibody, the methodfor treating, or the use according to (29), wherein the tauopathy isfrontotemporal lobar degeneration with tau pathology.

(32) The pharmaceutical composition, the anti-EphA4 antibody, the methodfor treating, or the use according to (31), wherein the frontotemporallobar degeneration with tau pathology is progressive supranuclear palsy,corticobasal degeneration, argyrophilic grain dementia, senile dementiaof the neurofibrillary tangle type, or Pick's disease.

(33) The pharmaceutical composition, the anti-EphA4 antibody, the methodfor treating, or the use according to (32), wherein the frontotemporallobar degeneration with tau pathology is progressive supranuclear palsy.

(34) The pharmaceutical composition, the anti-EphA4 antibody, the methodfor treating, or the use according to (32), wherein thefrontotemporallobar degeneration with tau pathology is corticobasaldegeneration.

(35) The pharmaceutical composition, the anti-EphA4 antibody, the methodfor treating, or the use according to (32), wherein the frontotemporallobar degeneration with tau pathology is argyrophilic grain dementia.

(36) The pharmaceutical composition, the anti-EphA4 antibody, the methodfor treating, or the use according to (32), wherein the frontotemporallobar degeneration with tau pathology is senile dementia of theneurofibrillary tangle type.

(37) The pharmaceutical composition, the anti-EphA4 antibody, the methodfor treating, or the use according to (32), wherein the frontotemporallobar degeneration with tau pathology is Pick's disease.

Effects of the Invention

The present disclosure provides an anti-EphA4 antibody that can bind toEphA4 and enhance the cleavage of EphA4, a nucleic acid that encodes theantibody, a vector that comprises the nucleic acid, a cell comprisingthe vector, a method of producing the antibody, and a pharmaceuticalcomposition comprising the antibody as the active ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the binding affinity of the anti-EphA4 monoclonal antibody(antibody A) against mouse and human EphA4.

FIG. 2 shows EphA4 cleavage enhancement activity of the anti-EphA4monoclonal antibody (antibody A) employing hippocampus neurons.

FIG. 3 shows mouse EphA4-mouse ligand binding inhibitory activity of theanti-EphA4 monoclonal antibody (antibody A).

FIG. 4 shows human EphA4-human ligand binding inhibitory activity of theanti-EphA4 monoclonal antibody (antibody A).

FIG. 5 shows the selectivity of the anti-EphA4 monoclonal antibody(antibody A) against each human Eph receptor.

FIG. 6 shows the selectivity of the anti-EphA4 monoclonal antibody(antibody A) against each mouse Eph receptor.

FIG. 7 shows the reactivity of the anti-EphA4 monoclonal antibody(antibody A) against mouse, rat, monkey, and human EphA4.

FIG. 8 shows the reactivity of the anti-EphA4 monoclonal antibody(antibody A) against human EphA4 extracellular region (ECD), ligandbinding domain (LBD), fibronectin type III domain 1 (FN1), andfibronectin type III domain 2 (FN2).

FIG. 9 shows the effect of the anti-EphA4 monoclonal antibody (antibodyA) on increasing the number of spines in the hippocampus neuron.

FIG. 10 shows the the effect to suppress tau phosphorylation of theanti-EphA4 monoclonal antibody in vivo.

FIG. 11A shows the amino acids of the EphA4-Ligand Binding Domain(EphA4-LBD) on the horizontal axis, and the structural region ofantibody A-Fab on the vertical axis. Black bits show the intersectingpoints of combinations where interaction is present.

FIG. 11B shows the surface structure of EphA4-Ligand Binding Domain(EphA4-LBD). In FIG. 11B, amino acid names and residue numbers containedin the binding region are shown at the corresponding positions, and theCDRs of H-chain and L-chain of the binding antibody A-Fab are shown in aribbon model.

FIG. 12 shows the affinity of the humanized anti-EphA4 monoclonalantibody (antibody B) against human EphA4.

FIG. 13 shows EphA4 cleavage enhancement activity of the humanizedanti-EphA4 monoclonal antibody (antibody B) in the hippocampus neuron.

FIG. 14 shows human EphA4-human ligand binding inhibitory activity ofthe humanized anti-EphA4 monoclonal antibody (antibody B).

FIG. 15 shows mouse EphA4-mouse ligand binding inhibitory activity ofthe humanized anti-EphA4 monoclonal antibody (antibody B).

FIG. 16 shows the selectivity of the humanized anti-EphA4 monoclonalantibody (antibody B) against human Eph receptor.

FIG. 17 shows the selectivity of the humanized anti-EphA4 monoclonalantibody (antibody B) against mouse Eph receptor.

FIG. 18 shows the reactivity of the humanized anti-EphA4 monoclonalantibody (antibody B) against mouse, rat, monkey, and human EphA4.

FIG. 19 shows the reactivity of the humanized anti-EphA4 monoclonalantibody (antibody B) against human EphA4 extracellular region (ECD),ligand binding domain (LBD), fibronectin type III domain 1 (FN1), andfibronectin type III domain 2 (FN2).

FIG. 20 shows the effect of the humanized anti-EphA4 monoclonal antibody(antibody B) on increasing the number of spines in the hippocampusneuron.

FIG. 21 shows human EphA4 cleavage enhancement activity of the humanizedanti-EphA4 monoclonal antibody (antibody B) in the hippocampus neuron.

FIG. 22 shows the effect of the humanized anti-EphA4 monoclonal antibody(antibody B) via MMP and ADAM on increasing the number of spines in thehippocampus neuron.

FIG. 23 shows the the effect to suppress tau phosphorylation of thehumanized anti-EphA4 monoclonal antibody (antibody B) in vivo.

DESCRIPTION OF EMBODIMENTS

The regions specified or coded by the SEQ ID NOs. used herein are asfollows:

Seq No. Sequence Region 1 Mouse EphA4 (amino acid sequence) 2Extracellular region of mouse EphA4 (amino acid sequence) 3 Mouse EphA4extracellular region-SEAP-His protein (amino acid sequence) 4 Signalsequence of mouse EphA4 (amino acid sequence) 5 Human EphA4 (amino acidsequence) 6 Extracellular region of human EphA4 (amino acid sequence) 7OligoDNA ad29S (nucleic acid sequence) 8 OligoDNA ad29AS (nucleic acidsequence) 9 5′ Forward primer (nucleic acid sequence) 10 3′ Reverseprimer for mouse IgG heavy chain (nucleic acid sequence) 11 3′ Reverseprimer for mouse Igκ light chain (nucleic acid sequence) 12 Heavy chainsignal sequence of antibody A (amino acid sequence) 13 Heavy chainvariable region of antibody A (amino acid sequence) 14 Light chainsignal sequence of antibody A (amino acid sequence) 15 Light chainvariable region of antibody A (amino acid sequence) 16 Heavy chainsignal sequence of antibody A (nucleic acid sequence) 17 Heavy chainvariable region of antibody A (nucleic acid sequence) 18 Light chainsignal sequence of antibody A (nucleic acid sequence) 19 Light chainvariable region of antibody A (nucleic acid sequence) 20 5′ Forwardprimer for antibody A heavy chain (nucleic acid sequence) 21 5′ Forwardprimer for antibody A light chain (nucleic acid sequence) 22 3′ Reverseprimer for antibody A heavy chain (nucleic acid sequence) 23 3′ Reverseprimer for antibody A light chain (nucleic acid sequence) 24 Heavy chainconstant region of antibody A (amino acid sequence) 25 Light chainconstant region of antibody A (amino acid sequence) 26 Heavy chain CDR1of antibody A (amino acid sequence) 27 Heavy chain CDR2 of antibody A(amino acid sequence) 28 Heavy chain CDR3 of antibody A (amino acidsequence) 29 Light chain CDR1 of antibody A (amino acid sequence) 30Light chain CDR2 of antibody A (amino acid sequence) 31 Light chain CDR3of antibody A (amino acid sequence) 32 Monkey EphA4 (amino acidsequence) 33 Extracellular region of monkey EphA4 (amino acid sequence)34 Signal sequence of human EphA4 (amino acid sequence) 35 Signalsequence of preprotrypsin (amino acid sequence) 36 Extracellular regionof human EphA4 (amino acid sequence) 37 Ligand binding domain of humanEphA4 (amino acid sequence) 38 Fibronectin type III domain 1 of humanEphA4 (amino acid sequence) 39 Fibronectin type III domain 2 of humanEphA4 (amino acid sequence) 40 Light chain FR IGKV1-17*01 of humanantibody (amino acid sequence) 41 Light chain FR JK4 of human antibody(amino acid sequence) 42 Heavy chain FR IGHV3-33*03 of human antibody(amino acid sequence) 43 Heavy chain FR JH6 of human antibody (aminoacid sequence) 44 Heavy chain CDR1 of antibody B (amino acid sequence)45 Heavy chain variable region HK2-42 of antibody B (amino acidsequence) 46 Light chain variable region L1-8 of antibody B (amino acidsequence) 47 Human IgG2 heavy chain constant region of antibody B (aminoacid sequence) 48 Human Igκ light chain constant region of antibody B(amino acid sequence) 49 Heavy chain CDR1 of antibody B (nucleic acidsequence) 50 Heavy chain CDR2 of antibody B (nucleic acid sequence) 51Heavy chain CDR3 of antibody B (nucleic acid sequence) 52 Light chainCDR1 of antibody B (nucleic acid sequence) 53 Light chain CDR2 ofantibody B (nucleic acid sequence) 54 Light chain CDR3 of antibody B(nucleic acid sequence) 55 Heavy chain variable region HK2-42 ofantibody B (nucleic acid sequence) 56 Light chain variable region L1-8of antibody B (nucleic acid sequence) 57 Human IgG₂ heavy chain constantregion of antibody B (nucleic acid sequence) 58 Human Igκ light chainconstant region of antibody B (nucleic acid sequence) 59 Heavy chainfull length sequence of antibody B (amino acid sequence) 60 Light chainfull length sequence of antibody B (amino acid sequence) 61 Heavy chainfull length sequence of antibody B (nucleic acid sequence) 62 Lightchain full length sequence of antibody B (nucleic acid sequence)

The present disclosure relates to an anti-EphA4 antibody that binds toEphA4.

The anti-EphA4 antibody according to the present disclosure is anantibody that can recognize and bind to EphA4, and as described below,the antibody may be an intact antibody, or may be a synthetic antibody(such as a recombinant antibody, a chimeric antibody, a humanizedantibody, and the like), as long as it possesses binding affinity withEphA4. EphA4 herein can be understood as referring to human-, mouse-,rat-, and monkey-derived EphA4. Human-, mouse-, rat-, and monkey-derivedEphA4 can be obtained from a public database where sequence informationis registered, such as Genbank provided by the United States NationalCenter for Biotechnology Information, or EphA4 gene sequence informationcan be obtained by designing primers based on the base sequenceinformation of EphA4 of a closely related animal specie, and thencloning from RNA extracted from the desired animal specie. For example,the base sequence information of human, mouse, rat, and monkey EphA4 isregistered in the database as Genbank Accession Nos. NM_004438.5,NM_007936.3, NM_001162411.1, and NM_001260870.1, respectively.

In one aspect, the anti-EphA4 antibody is an antibody that specificallybinds to EphA4. The term “specific binding” is a term well-known tothose skilled in the pertaining technical field, and methods fordetermining the specific binding between an antibody or an antigenbinding fragment thereof and an antigen or an epitope are alsowell-known. In one embodiment, “specific binding” is understood as thatthe anti-EphA4 antibody can bind to EphA4 by immunological reaction withhigher binding affinity and binding activity, more rapidly, and/or for alonger period of time compared to when binding with other targetmolecules. This does not mean that the antibody that specifically bindsto EphA4 does not bind to other target molecules. In another embodiment,“specific binding” may be shown by an antibody having a KD against EphA4of at least about 10⁻⁷ M, or at least about 10⁻⁸ M, or at least about10⁻⁹ M, or lower. Moreover, in another further embodiment, “specificbinding” is understood as binding to EphA4 by immunological reaction butdoes not substantially bind to other family molecules of the Ephreceptor.

In one aspect, the anti-EphA4 antibody is an antibody that binds to theextracellular region of EphA4. In one embodiment, the anti-EphA4antibody is an antibody that binds to the ligand binding domain (LBD)among the extracellular regions of EphA4.

In one embodiment, the anti-EphA4 antibody can specifically bind toEphA4 and enhance the cleavage of EphA4. In a particular embodiment, theanti-EphA4 antibody can specifically bind to EphA4 and enhance thecleavage of the EphA4 extracellular domain by matrix metalloprotease(MMP) or ADAM (a disintegrin and metalloproteinase).

In one embodiment, the anti-EphA4 antibody can specifically bind toEphA4 and inhibit the binding between EphA4 and ephrin which is a ligandthereof.

In another embodiment, the anti-EphA4 antibody can specifically bind toEphA4 and increase the number of spines in the hippocampus neuron orstabilize the spines in the hippocampus neuron.

In one embodiment, the present disclosure encompasses an anti-EphA4antibody that can specifically bind to at least one of human EphA4,mouse EphA4, rat EphA4, and monkey EphA4 and inhibit the binding with aligand thereof. In another embodiment, the present disclosureencompasses an anti-EphA4 antibody that can specifically bind to two ormore of human EphA4, mouse EphA4, rat EphA4, and monkey EphA4 andinhibit the binding with a ligand thereof. In another furtherembodiment, the present disclosure encompasses an anti-EphA4 antibodythat can specifically bind to all of human EphA4, mouse EphA4, ratEphA4, and monkey EphA4 and inhibit the binding with a ligand thereof.

For the method for measuring the antigen binding property (such asbinding affinity and cross-species reactivity) of the anti-EphA4antibody, methods well-known to those skilled in the art in thepertaining technical field may be employed. For example, bindingaffinity may be measured using, but not limited to, Biacore™ biosensor,KinExA biosensor, scintillation proximity assay, ELISA, ORIGENimmunoassay (IGEN), flow cytometry, fluorescence quenching, fluorescencemetastasis, yeast display, and/or immunostaining. The neutralizingactivity of anti-EphA4 antibody against the binding between EphA4 and aligand thereof may be measured using, but not limited to, Biacore™biosensor, ELISA, and/or flow cytometry.

The anti-EphA4 antibody according to the present disclosure may be amonoclonal antibody as long as it binds to EphA4.

The anti-EphA4 antibody according to the present disclosure may be ofany class such as IgG, IgA, or IgM (or subclasses thereof), and is notlimited to a particular class. Immunoglobulins are classified intodifferent classes depending on the antibody amino acid sequence of theconstant region of the heavy chain (may be referred to as H-chain).There are five major immunoglobulin classes: IgA, IgD, IgE, IgG, andIgM, and some of these may be further subdivided into subclasses(isotypes) of e.g. IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The constantregions of the heavy chain corresponding to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. Moreover,the types of the light chain (may be referred to as L-chain) of theantibody are the λ chain and the κ chain. The anti-EphA4 antibodyaccording to the present disclosure may be an IgG antibody, for examplemay be an IgG₁ antibody or an IgG₂ antibody, and the like. Moreover, theanti-EphA4 antibody according to the present disclosure may be in someinstances in the form of a monomer, a dimer, or a multimer.

The variable region of the antibody according to the present disclosuremay mean the variable region of the antibody light chain and/or thevariable region of the antibody heavy chain, and the constant region ofthe antibody may mean the constant region of the antibody light chainand/or the constant region of the antibody heavy chain. The variableregions of the heavy and light chains each consist of four frameworkregions (FRs) connected by three CDRs also known ascomplementarity-determining regions. The CDRs in each chain are kept inproximity by FRs, and along with the CDRs in the other chain, contributeto the formation of the antigen binding site of the antibody.Technologies for determining CDRs include, but are not limited to, forexample, (1) an approach based on cross-species sequence variability(e.g. Kabat et al, Sequences of Proteins of Immunological Interest, 5thed., 1991, National Institutes of Health, Bethesda Md.); and (2) anapproach based on crystal structure research of antigen-antibodycomplexes (Al-lazikani et al., 1997 J. Molec. Biol. 273: 927-948). Theseand other approaches may be employed in combination.

A monoclonal antibody here in may mean an antibody obtained from apopulation of essentially uniform antibodies. In other words, individualantibodies contained in the population is identical except for naturalmutants that may possibly be present in small amounts. Monoclonalantibodies are against single antigenic sites, and are very specific.Further, in contrast to a typical polyclonal antibody which targetsdifferent antigens or different epitopes, each monoclonal antibodytargets a single epitope of an antigen. The modifier “monoclonal”indicates the property of an antibody obtained from an essentiallyuniform antibody population, and is not to be understood in a limitedway as requiring the production of the antibody by a particular method.

The anti-EphA4 antibody according to the present disclosure may be amouse antibody, a chimeric antibody, or a humanized antibody. A chimericantibody is e.g. an antibody where the variable region of a non-human(such as a mouse or a rat) antibody is fused with the constant region ofa human antibody, and for example may refer to an antibody where thevariable region is derived from a non-human antibody and the constantregion is derived from a human antibody. A humanized antibody is e.g. anantibody where a complementarity-determining region (CDR (may also bereferred to as a hypervariable region)) of a non-human antibody isintroduced into a human antibody, and for example may refer to anantibody where the CDR is derived from a non-human antibody and theremaining regions of the antibody are derived from a human antibody.Note that the borderline between a chimeric antibody and a humanizedantibody does not necessarily need to be clear, and an antibody may bein a state that may be called a chimeric antibody or a humanizedantibody. Moreover, in a chimeric antibody or a humanized antibody, theantibody region derived from a human antibody (FR, constant region) doesnot necessarily need to be all composed of amino acids derived from ahuman antibody, and may comprise one or multiple amino acids derivedfrom a non-human antibody, as long as it can be used normally in a humansubject. One embodiment of a humanized antibody is an antibody where theCDR is derived from a rodent antibody and the remaining regions of theantibody are derived from a human antibody. A particular embodiment of ahumanized antibody is an antibody where the CDR is derived from a mouseantibody and the remaining regions of the antibody are derived from ahuman antibody. In these embodiments, the CDR may comprise one ormultiple amino acids derived from a non-rodent antibody or one ormultiple amino acids derived from a non-mouse antibody, and the antibodyregions other than the CDR may comprise one or multiple amino acidsderived from a non-human antibody. Here, “multiple” is, but it notlimited to, 2-20, or 2-15, such as 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,3, or 2, or within 10%, within 9%, within 8%, within 7%, within 6%,within 5%, within 4%, within 3%, within 2%, or within 1% of the numberof amino acids in an amino acid sequence. Humanization can be performedwith a CDR transplantation method (Kontermann and Dubel, AntibodyEngineering, Springer Lab Manual (2001) and Tsurushita et al., Methods36: 69-83 (2005)), and further may also be performed by substituting theCDR sequence with a corresponding sequence in the human antibody withmethods well-known in the pertaining technical field (see e.g. Jones etal., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-327(1988); and Verhoeyen et al., Science 239: 1534-1536 (1988)).

In order to decrease antigenicity, it may be important to select the useof human variable regions in both light and heavy chains uponpreparation of the humanized antibody. According to the “best-fit”method, the variable region sequence of the rodent antibody is screenedagainst the entire library of known human FR sequences. Following that,a human sequence that is the closest to the rodent sequence is acceptedas the human FR of the humanized antibody. See e.g. Sims et al., J.Immunol. 151: 2296-2308 (1993) and Chothia et al., J. Mol. Biol. 196:901-917 (1987). In another method, a particular framework derived from asequence common in all human antibodies of a particular subgroup of thelight or heavy chain is employed. The same frame work may be employedfor several different humanized antibodies. See e.g. Carter et al.,Proc. Natl. Acad. Set USA 89: 4285-4289 (1992) and Presta et al., J.Immunol. 151: 2623-2632 (1993).

Further, in general, it is desirable that the humanized antibody retainsthe high binding affinity towards the antigen and other preferredbiological properties. To this end, according to one method, humanizedantibodies are prepared by an analysis step of the parent sequence andvarious conceptual humanized products employing three-dimensional modelsof the parent and humanized sequences. In general, three-dimensionalimmunoglobulin models are available and known to those skilled in theart. Computer programs that illustrate and present promisingthree-dimensional conformations of selected candidate immunoglobulinsequences are available. Investigation of these presentations will allowanalysis of possible roles of residues in the functions of candidateimmunoglobulin sequences, i.e. analysis of residues that influence theability of candidate immunoglobulins to bind to their antigens. Withthis method, FR residues can be selected from recipient and importedsequences and used in combination so that desirable antibody propertiessuch as increase in the binding affinity to single or multiple targetantigen(s) (such as EphA4 or a fragment thereof) are accomplished.

Needless to say, an antibody with appropriate alteration (such asmodification of the antibody, or partial substitution, addition and/ordeletion of the amino acid sequence of the antibody) in the chimeric orhumanized antibody exemplified above while retaining the function of theantibody (or in order to add or improve the function of the antibody) isalso encompassed in the anti-EphA4 antibody according to the presentdisclosure. More specifically, an antibody having alteration in theamino acid sequence of the constant region in order to modify theeffector function of the antibody is also included in the scope of thepresent disclosure. For example, an antibody having valine (Val) atposition 234 (Eu numbering) of human IgG₂ antibody substituted toalanine (Ala) and having glycine (Gly) at position 237 substituted toalanine (Ala) in order to reduce antibody-dependent cellularcytotoxicity (ADCC) activity and/or antibody-dependent cellularphagocytosis (ADCP) activity etc. is also included in the scope of thepresent disclosure. Further, a bispecific antibody that has an antibodybinding site having the CDR sequence of the anti-EphA4 antibodyaccording to the present disclosure together with an antigen bindingsite that binds to a different antigen (Kontermann (2012), mAbs 4,182-97) is also included in the scope of the present disclosure.

The anti-EphA4 antibody according to the present disclosure may bemodified as desired. Modification of the anti-EphA4 antibody may be amodification that changes (a) the three-dimensional structure of theamino acid sequence in the region to be modified, such as sheet or helixconformation etc.; (b) the charge or hydrophobic state of the moleculeat the target site; or (c) the effect of the modification on themaintenance of the side chain volume, or may be a modification in whichthese changes are not clearly observed.

Modification of the anti-EphA4 antibody according to the presentdisclosure may be accomplished by e.g. substitution, deletion, addition,and the like of the constituent amino acid residues.

An amino acid herein is employed in its broadest meaning, and includesnot only natural amino acids, such as serine (Ser), asparagine (Asn),valine (Val), leucine (Leu), isoleucine (Ile), alanine (Ala), tyrosine(Tyr), glycine (Gly), lysine (Lys), arginine (Arg), histidine (His),aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), threonine(Thr), cysteine (Cys), methionine (Met), phenylalanine (Phe), tryptophan(Trp), and proline (Pro), but also non-natural amino acids such as aminoacid variants and derivatives. Those skilled in the art will naturallyunderstand in light of this broad definition that e.g. L-amino acids;D-amino acids; chemical modified amino acids such as amino acid variantsand amino acid derivatives; amino acids that will not be proteincomponents in the body such as norleucine, β-alanine, and ornithine; andchemically synthesized compounds having amino acid properties well-knownto those skilled in the art, and the like are included as the aminoacids of the present specification. Examples of a non-natural amino acidcan include, e.g., α-methylamino acids (such as α-methylalanine),D-amino acids (such as D-aspartic acid and D-glutamic acid),histidine-like amino acids (such as 2-amino-histidine,β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine, andα-methyl-histidine), amino acids having excess methylene on the sidechain (“homo” amino acid), and amino acids where the carboxylatefunctional group amino acid in the side chain is substituted with asulfonate group (such as cysteic acid).

Naturally-occurring amino acid residues may be classified into e.g.groups below based on general side chain properties:

(1) Hydrophobic: Met, Ala, Val, Leu, Ile;

(2) Neutral hydrophilic: Asn, Gln, Cys, Ser, Thr;

(3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg;

(5) Residues that influence chain orientation: Gly, Pro; and

(6) Aromatic: Trp, Tyr, Phe.

Nonconservative substitution of the amino acid sequence constituting theantibody may be performed by exchanging an amino acid belonging to oneof these groups with an amino acid belonging to another group. A moreconservative substitution may be performed by exchanging an amino acidbelonging to one of these groups with another amino acid in the samegroup. Similarly, deletion or substitution of the amino acid sequencemay be appropriately performed.

Modification of amino acids configuring the antibody may be e.g.glycosylation by a carbohydrate or post-translational modifications suchas acetylation or phosphorylation. The antibody may be glycosylated at aconserved position in its constant region. Glycosylation of an antibodyis ordinarily either N-linked or O-linked. N-linked means the binding ofa carbohydrate portion to the side chain of an asparagine residue.Tripeptide sequences asparagine-X-serine, asparagine-X-threonine, andasparagine-X-cysteine (wherein X is any amino acid other than proline)are recognition sequences for enzymatically adding the carbohydrateportion to the asparagine side chain. When one of these tripeptidesequences is present in the antibody, a potential glycosylation site ispresent. O-linked glycosylation may be the binding of eitherN-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid(such as serine or threonine), and in some instances may be the bindingto 5-hydroxyproline or 5-hydroxylysine. Those skilled in the art canappropriately select the glycosylation condition (such as the type ofhost cell or cell medium and pH etc., when glycosylation is performedwith a biological means) according to the objective.

The anti-EphA4 antibody according to the present disclosure may furtherbe modified by other modification methods, alone or in combination,based on common technical knowledge well-known to those skilled in theart.

The anti-EphA4 antibody according to the present disclosure can beproduced by a method well-known to those skilled in the art. Forexample, the antibody may be produced by integrating the nucleic acidencoding the anti-EphA4 antibody according to the present disclosureinto an expression vector, introducing the expression vector into a hostcell, and culturing the host cell. Accordingly, the present disclosureencompasses a nucleic acid encoding the anti-EphA4 antibody, a vectorthat comprises the nucleic acid, a host cell comprising the vector, anda method of producing the anti-EphA4 antibody comprising a step ofculturing the host cell.

The nucleic acid encoding the anti-EphA4 antibody according to thepresent disclosure may have DNA encoding a signal sequence, or may haveDNA encoding a signal sequence at the 5′ terminal of the DNA encodingthe heavy chain variable region and the DNA encoding the light chainvariable region. A signal sequence is amino acid residues present at theN-terminal of the protein that is necessary for a secretory protein oran integral membrane protein to pass through the lipid bilayer afterbeing synthesized on a ribosome, and in the present disclosure, is notparticularly limited as long as it is a sequence having this function.Signal sequences that may be contained in the anti-EphA4 antibodyaccording to the present disclosure include signal sequences derivedfrom human, mouse, rat, rabbit, donkey, goat, horse, bird, dog, cat,yeast, and the like. Specifically, in the present disclosure, a peptidecomprising an amino acid sequence represented by SEQ ID NO. 12 or 16 canbe included as a signal sequence related to the heavy chain, and apeptide comprising an amino acid sequence represented by SEQ ID NO. 14or 18 can be included as a signal sequence related to the light chain.Moreover, as long as it is functionally equivalent, the signal sequencemay have substitution, addition, and/or deletion of one or multiple(such as 2, 3, 4, or 5) amino acids in the amino acid sequencerepresented by SEQ ID NO. 12 or 16 and the amino acid sequencerepresented by SEQ ID NO. 14 or 18.

The anti-EphA4 antibody according to the present disclosure may be thatisolated or purified according to a method well-known to those skilledin the art.

“Isolated” or “purified” herein means artificially isolated or purifiedfrom the natural state. If the molecule or composition is naturallyoccurring, it is “isolated” or “purified” when it is changed or removedfrom the original environment, or both. Examples of an isolation orpurification method include, but are not limited to, means byelectrophoresis, molecular biology, immunology, or chromatography, andthe like, specifically, ion exchange chromatography, hydrophobicchromatography, reverse phase HPLC chromatography, isoelectric focusing,or alkali extraction method, and the like.

In one embodiment, the anti-EphA4 antibody comprises the following CDRs:

(a) a heavy chain CDR1 consisting of the amino acid sequence shown inSEQ ID NO. 44;(b) a heavy chain CDR2 consisting of the amino acid sequence shown inSEQ ID NO. 27;(c) a heavy chain CDR3 consisting of the amino acid sequence shown inSEQ ID NO. 28;(d) a light chain CDR1 consisting of the amino acid sequence shown inSEQ ID NO. 29;(e) a light chain CDR2 consisting of the amino acid sequence shown inSEQ ID NO. 30; and(f) a light chain CDR3 consisting of the amino acid sequence shown inSEQ ID NO. 31.

In one embodiment, the anti-EphA4 antibody is a humanized antibody or achimeric antibody, and in a particular embodiment a humanized antibody.

In another embodiment, the anti-EphA4 antibody comprises heavy and lightchains, the variable region of the heavy chain comprises the amino acidsequence shown in SEQ ID NO. 45, and the variable region of the lightchain comprises the amino acid sequence shown in SEQ ID NO. 46. Notethat in the embodiment, the variable region of the heavy chain and/orthe variable region of the light chain may comprise an amino acidsequence having one or multiple aminoacids substituted, added, and/ordeleted in the amino acid sequence shown in SEQ ID NO. 45 and/or theamino acid sequence shown in SEQ ID NO. 46. Here, “multiple” is notlimited as long as it retains the binding affinity towards EphA4 andenhances the cleavage of EphA4, and is 2-15, or 2-10, such as 9, 8, 7,6, 5, 4, 3, or 2, or within 10%, such as within 9%, within 8%, within7%, within 6%, within 5%, within 4%, within 3%, within 2%, or within 1%of the number of amino acids in an amino acid sequence.

In one embodiment, the heavy chain of the anti-EphA4 antibody comprisesthe constant region of human IgG₂.

In a particular embodiment, the constant region of human IgG₂ comprisesthe amino acid sequence of SEQ ID NO. 47.

In one embodiment, the light chain of the anti-EphA4 antibody comprisesthe constant region of human Igκ.

In a particular embodiment, the constant region of human Igκ comprisesthe amino acid sequence of SEQ ID NO. 48.

In one embodiment, the anti-EphA4 antibody comprises a heavy chaincomprising the amino acid sequence shown in SEQ ID NO. 59 and a lightchain comprising the amino acid sequence shown in SEQ ID NO. 60.

In another embodiment, for example, for reasons such as decreasing theununiformity of antibodies produced by antibody-producing cells (U.S.Patent Application Publication No. 2010/0297697 or Liu H et al., MAbs.2014 September-October; 6 (5): 1145-1154), the anti-EphA4 antibody haslysine positioned at the C-terminal (carboxy terminal) of the heavychain deleted. In the present disclosure, an anti-EphA4 antibody havingthe C-terminal lysine of the heavy chain deleted also includes ananti-EphA4 antibody having the C-terminal lysine of the heavy chaindeleted by genetic modification or an anti-EphA4 antibody having theC-terminal lysine of the heavy chain cleaved post-translationally bycarboxypeptidase etc., and the like. Moreover, in the presentdisclosure, an anti-EphA4 antibody having the C-terminal lysine of theheavy chain deleted includes not only an anti-EphA4 antibody having theC-terminal lysine deleted in both heavy chains, but also an anti-EphA4antibody having the C-terminal lysine deleted in only one heavy chain.

In one aspect, the present disclosure relates to an isolated nucleicacid encoding an anti-EphA4 antibody. An isolated nucleic acid encodingan anti-EphA4 antibody refers to one or more nucleic acid moleculesencoding the heavy chain and/or light chain of an anti-EphA4 antibody.In one embodiment, the nucleic acid according to the present disclosureencodes the heavy chain of the anti-EphA4 antibody. In anotherembodiment, the nucleic acid according to the present disclosure encodesthe light chain of the anti-EphA4 antibody. In another furtherembodiment, the nucleic acid according to the present disclosure encodesthe heavy and light chains of the anti-EphA4 antibody. The nucleic acidaccording to the present disclosure also includes a first nucleic acidmolecule encoding the heavy chain of the anti-EphA4 antibody and asecond nucleic acid molecule encoding the light chain of the anti-EphA4antibody.

In another aspect, the present disclosure relates to a vector comprisingthe isolated nucleic acid encoding the anti-EphA4 antibody. The vectoraccording to the present disclosure refers to one or more vectorscomprising the isolated nucleic acid encoding the anti-EphA4 antibody.In one embodiment, the vector according to the present disclosure is avector comprising the nucleic acid encoding the heavy chain of theanti-EphA4 antibody and the nucleic acid encoding the light chain of theanti-EphA4 antibody. In another embodiment, the vector according to thepresent disclosure is a vector comprising the nucleic acid encoding theheavy and light chains of the anti-EphA4 antibody. In another furtherembodiment, the vector according to the present disclosure comprises afirst vector comprising the nucleic acid encoding the heavy chain of theanti-EphA4 antibody and a second vector comprising the nucleic acidencoding the light chain of the anti-EphA4 antibody. The vectoraccording to the present disclosure may be, but not limited to, aplasmid, a cosmid, a virus, a phage, and the like. For example, as aviral vector, retroviral, lentiviral, adenoviral, adeno-associatedviral, or herpes simplex viral vector, and the like are also included inthe vector according to the present disclosure.

In yet another aspect, a host cell comprising the vector according tothe present disclosure, and a method of producing an anti-EphA4 antibodycomprising a step of culturing the host cell are also included in thepresent disclosure. The host cell according to the present disclosuremay be, but not limited to, E. coli cells, monkey COS cells, Chinesehamster ovary (CHO) cells, NS0 cells, and the like. In one embodiment,the method of producing an anti-EphA4 antibody comprises a step ofculturing the host cell, and a step of recovering the anti-EphA4antibody secreted from the host cell (or culture medium of host cell).

In one aspect, the present disclosure relates to a pharmaceuticalcomposition comprising an anti-EphA4 antibody. The pharmaceuticalcomposition according to the present disclosure can be manufacturedaccording to known methods such as methods described in the Pharmacopeiaof Japan (JP), the United States Pharmacopeia (USP), or the EuropeanPharmacopeia (EP), and the like.

The anti-EphA4 antibody according to the present disclosure may beuseful for treating Alzheimer's disease. In other words, in otheraspects, the present disclosure encompasses a method for treatingAlzheimer's disease comprising a step of administering a therapeuticallyeffective amount of an anti-EphA4 antibody to a subject havingAlzheimer's disease. Moreover, in other aspects, the present disclosureencompasses the use of an anti-EphA4 antibody for manufacturing atherapeutic drug for Alzheimer's disease. In other aspects, the presentdisclosure encompasses an anti-EphA4 antibody for use in the treatmentof Alzheimer's disease.

The anti-EphA4 antibody according to the present disclosure may beuseful for treating tauopathy. In other words, in other aspects, thepresent disclosure encompasses a method for treating tauopathycomprising a step of administering a therapeutically effective amount ofan anti-EphA4 antibody to a subject having tauopathy. Moreover, in otheraspects, the present disclosure encompasses the use of an anti-EphA4antibody for manufacturing a therapeutic drug for tauopathy. In otheraspects, the present disclosure encompasses an anti-EphA4 antibody foruse in the treatment of tauopathy. Tauopathy of the present disclosureincludes Alzheimer's disease or frontotemporal lobar degeneration withtau pathology (FTLD-tau). Moreover, frontotemporal lobar degenerationwith tau pathology includes progressive supranuclear palsy (PSP),corticobasal degeneration (CBD), argyrophilic grain dementia (AGD),senile dementia of the neurofibrillary tangle type (SD-NFT), Pick'sdisease (PiD), and the like.

The anti-EphA4 antibody according to the present disclosure can beemployed in the therapeutic method alone or in combination with otheragents or compositions. For example, the anti-EphA4 antibody accordingto the present disclosure may be administered at the same or differenttimes as another agent. Such combination therapy includes combinationadministration (two or more agents are included in the same or separateformulation) and separate administration (such as simultaneous orsequential). When two or more agents are separately administered, theadministration of the anti-EphA4 antibody according to the presentdisclosure may be performed before or after the accompanying therapeuticmethod.

The subject for administering the pharmaceutical composition accordingto the present disclosure is not limited, and can be employed for e.g. ahuman or non-human mammal (such as monkey, mouse, rat, rabbit, cow,horse, and goat).

The method of administering the pharmaceutical composition according tothe present disclosure to a subject (such as administration route,dosage, the number of administrations per day, and administrationtiming) is not limited, and can be appropriately decided by thoseskilled in the art (such as a physician) according to the health stateof the subject, the extent of the disease, the type of agents used incombination, and the like.

Those skilled in the art shall recognize that as long as it is nottechnically contradicting, the present invention may be carried out inan appropriate combination of any one or more of any and all aspectsdescribed herein. Further, those skilled in the art shall recognize thatas long as it is not technically contradicting, it is preferred that thepresent invention is carried out in an appropriate combination of anyand all preferred or advantageous aspects described herein.

All of the disclosures of the literatures cited herein shall be regardedas clearly cited herein by reference, and those skilled in the art canunderstand the related disclosure contents in these literatures byciting them as a part of the present specification according to thecontext herein without departing from the spirit and scope of thepresent invention.

The literatures cited herein are provided with the sole purpose ofdisclosing the related technology preceding the filing date of thepresent application, and are not to be construed as an admission by thepresent inventors that the present invention does not hold the right toprecede said disclosures due to prior inventions or for any otherreason. All of the description of all these literatures is based on theinformation available to the present applicants, and does not configurein any way an acknowledgement that these description contents areaccurate.

The terms used herein are employed for describing particularembodiments, and do not intend to limit the invention.

The term “comprise” as used herein, unless the content clearly indicatesto be understood otherwise, intends the presence of the described items(such as components, steps, elements, or numbers), and does not excludethe presence of other items (such as components, steps, elements, andnumbers). The term “consist of” encompasses the aspects described by theterms “consist of” and/or “consist essentially of.”

The term “neutralizing activity” as used herein means the activity toinhibit the binding between EphA4 and a ligand thereof, and/or theactivity to inhibit signal transduction or molecular expression responseor functionality change of cells which are induced by EphA4 due tobinding to a ligand thereof in the human body.

Unless otherwise defined, all terms used herein (including technical andscientific terms) have the same meanings as those broadly recognized bythose skilled in the art of the technology to which the presentinvention belongs. The terms used herein, unless explicitly definedotherwise, are to be construed as having meanings consistent with themeanings herein and in related technical fields, and shall not beconstrued as having idealized or excessively formal meanings.

Terms such as first and second are employed to express various elements,and it should be recognized that these elements are not to be limited bythese terms per se. These terms are employed solely for the purpose ofdiscriminating one element from another, and it is for example possibleto describe a first element as a second element, and similarly, todescribe a second element as a first element without departing from thescope of the present invention.

The numeric values employed herein for indicating component content ornumeric value range and the like, unless explicitly indicated, are to beunderstood as being modified by the term “about.” For example, “4° C.,”unless explicitly indicated, is understood to mean “about 4° C.,” and itis obvious that those skilled in the art can rationally understand theextent thereof in concordance with technical common sense and themeaning of the passages herein.

It should be recognized that unless clearly indicated to mean otherwisein context, when used in the specification and the claims herein, eachaspect represented in singular form may also be a plural form as long asit is not technically contradicting, and vice versa.

The present invention will now be described in further detail withreference to Examples. However, the present invention can be embodied byvarious aspects, and shall not be construed as being limited to theExamples described herein. Those skilled in the art of related technicalfields will be able to carry out the present invention with variousmodifications, additions, deletions, substitutions, and the like withoutaltering the spirit or scope of the present invention.

EXAMPLES Reference Example 1: Preparation of Anti-EphA4 MonoclonalAntibody (A) Preparation of Mouse Anti-EphA4 Monoclonal Antibody

In order to prepare a monoclonal antibody that binds to mouse EphA4(Genbank Accession No. NP_031962.2, SEQ ID NO. 1), a protein havingsecretory alkaline phosphatase (SEAP) and histidine tag fused to theextracellular region of mouse EphA4 (positions 20-547) (SEQ ID NO. 2)(hereinafter referred to as “mouse EphA4 extracellular region-SEAP-Hisprotein,” SEQ ID NO. 3) was prepared by the following steps.

First, the DNA sequences encoding the signal sequence (SEQ ID NO. 4) andthe extracellular region (SEQ ID NO. 2) of mouse EphA4 were amplified byRT-PCR with total RNA derived from mouse brain, and cloned into the SalI/Not I site of pENTR1A vector (Invitrogen/LifeTechnologies) having theDNA sequence encoding SEAP and histidine tag. Next, the DNA sequenceencoding the signal sequence and extracellular region of mouse EphA4,SEAP, and histidine tag was transferred to pcDNA3.1_rfcB vector by LRreaction of Gateway System (Invitrogen/LifeTechnologies) to constructpcDNA 3.1-mouse EphA4 extracellular region-SEAP-His expression vector.The constructed pcDNA 3.1-mouse EphA4 extracellular region-SEAP-Hisexpression vector was transfected into HEK293 EBNA cells(Invitrogen/LifeTechnologies) with TransIT-LT1 (TAKARA). After 6 days ofincubation (5% CO₂, 37° C.), the culture supernatant was recovered. Fromthe recovered culture supernatant, mouse EphA4 extracellularregion-SEAP-His protein (SEQ ID NO. 3) was purified with Protino column(MACHEREY-NAGEL).

Twenty micrograms of mouse EphA4 extracellular region-SEAP-His proteinwas mixed with the same amount of TiterMax Gold adjuvant (TiterMax USA)or GERBU adjuvant (GERBU Biotechnik GmbH), and subcutaneously injectedinto the footpad of Balb/c mice. Mouse EphA4 extracellularregion-SEAP-His protein was then similarly administered on Days 3, 7,and 10. Here, TiterMax Gold adjuvant (TiterMax USA) was used only on Day10, and GERBU adjuvant (GERBU Biotechnik GmbH) was used on Days 3, 7,and 10. Mice were sacrificed on Day 13, and peripheral lymph nodes wererecovered to prepare lymph node cells. The prepared lymph node cells andP3U1 myeloma cells (endowed from Kyoto University) were fused at aproportion of 5:1 in the presence of GenomeONE-CF (Ishihara SangyoKaisha, Ltd.). The fused cells were cultured in a 96-well plastic plate.After 7 days of incubation (5% CO₂, 37° C.), the culture supernatant wasrecovered.

Employing the culture supernatant obtained, wells having reactivityagainst mouse, rat, and human EphA4 were picked up.

Reactivity against mouse, rat, and human EphA4 was evaluated with ELISAwith proteins having the Fc region of human IgG₁ and histidine tag fusedto the extracellular region of mouse EphA4, the extracellular region(positions 20-547) of rat EphA4 (Genbank Accession No. NP_001155883.1),or the extracellular region (positions 20-547) (SEQ ID NO. 6) of humanEphA4 (Genbank Accession No. NP_004429.1, SEQ ID NO. 5) (hereinafterreferred to “mouse EphA4 extracellular region-Fc-His protein,” “ratEphA4 extracellular region-Fc-His protein,” or “human EphA4extracellular region-Fc-His protein,” respectively).

Mouse, rat, or human EphA4 extracellular region-Fc-His proteins wereprepared by the following steps. Initially, pcDNA 3.1-mouse, rat, orhuman EphA4 extracellular region-Fc-His expression vectors wereconstructed. First, the DNA sequences encoding the signal sequence andthe extracellular region of mouse, rat, or human EphA4 were amplified byRT-PCR with total RNA derived from mouse, rat, or human brain, andcloned into the Sal I/Not I site of pENTRIA vector(Invitrogen/LifeTechnologies) having the DNA sequence encoding Fc andhistidine tag. Next, the DNA sequences encoding the signal sequence andextracellular region of mouse, rat, or human EphA4, Fc, and histidinetag were transferred to pcDNA 3.1_rfcB vector by LR reaction of GatewaySystem (Invitrogen/LifeTechnologies) to construct pcDNA 3.1-mouse, rat,or human EphA4 extracellular region-Fc-His expression vectors. Theseexpression vectors constructed were transfected into HEK293 EBNA cells(Invitrogen/LifeTechnologies) with TransIT-LT1 (TAKARA). After 6 days ofincubation (5% CO₂, 37° C.), the culture supernatant was recovered.

ELISA employing mouse, rat, or human EphA4 extracellular region-Fc-Hisproteins was performed following the steps below. Anti-human IgGantibody (Jackson ImmunoResearch Laboratories) was coated onto the wellsof a 96-well plate (Nunc). After incubating at 4° C. overnight, wellswere blocked at room temperature for one hour with 1×Block ACE(Dainippon Seiyaku). After washing three times with 0.02 Tween 20/PBS(Nacalai Tesque), a culture supernatant comprising mouse, rat, or humanEphA4 extracellular region-Fc-His protein was added to each well (finalconcentration 1 nM), and this was incubated at room temperature for onehour. After washing three times, the culture supernatant of the fusedcells was added to each well. After incubating at room temperature forone hour and washing three times, horseradish peroxidase-labeledanti-mouse IgG antibody (Jackson ImmunoResearch Laboratories) was added,and this was incubated at room temperature for one hour. After washingthree times, TMBZ (3,3′,5,5′-tetramethylbenzidine, Sigma) solution wasadded to each well, and this was incubated at room temperature for 5-20minutes. An equal amount of the stop solution (2 N H₂SO₄, Wako PureChemical) was added to each well, and absorbance at 450 nm was read witha microplate reader (PerkinElmer).

Hybridomas were cloned from the wells picked up through the above stepsby limiting dilution method, and hybridoma clones expressing mouseanti-EphA4 antibody having binding activity against mouse, rat, andhuman EphA4 was ultimately obtained.

The hybridoma clones obtained were cultured, and mouse anti-EphA4monoclonal antibody was purified from the culture supernatant withProtein A (GE Healthcare).

(B) Evaluation of EphA4 Cleavage Enhancement Activity

Preparation of rat hippocampus neurons was performed following the stepsbelow. Fetuses were taken out of a rat at Day 18 of pregnancy (CharlesRiver Laboratories Japan), and the head was cut open to take out thebrain. The hippocampus region was cut out under a stereomicroscope,placed in the digestion solution (137 mM NaCl (Wako Pure Chemical), 5 mMKCl (Wako Pure Chemical), 7 mM Na₂HPO₄ (Wako Pure Chemical), 25 mM Hepes(DOJINDO), 0.5 mg/mL DNase (Sigma), and 0.25% trypsin (Lifetechnologies)), and shaken at 37° C. for 10 minutes. The solution wasremoved, and 20S Fetal bovine serum/Hanks buffer (Sigma) was added.After removing the solution and washing twice in Hanks buffer, thehippocampus tissue was pipetted in Hanks buffer to prepare a cellsuspension. Cells were seeded in a 96-well dish (Falcon) coated withpoly-L-lysine containing culture fluid (Neurobasal medium (Lifetechnologies), 1×B-27 supplement (Life technologies), and 0.5 mML-glutamine (Life technologies)).

Evaluation of EphA4 cleavage enhancement activity employing hippocampusneurons was performed following the steps below. Rat hippocampus neuronsseeded in a 96-well dish (Falcon) were treated with anti-EphA4monoclonal antibody (67 nM) and γ selectase inhibitory drug Compound E(50 nM, Enzo Life Sciences). Sixteen hours later, this was washed withPBS (Wako Pure Chemical), SDS sample buffer (Laemmli sample buffer(Bio-Rad) and 5% 2-mercaptoethanol (Bio-Rad)) was added to recover thecells, and this was boiled for 5 minutes. SDS-PAGE was performed withthis sample, western blotting with anti-EphA4 monoclonal antibody(Abnova) was performed, the band strength was quantified, and the valueof EphA4 C-terminal fragment/full length EphA4 was calculated.

Mouse anti-EphA4 monoclonal antibody having activity that enhances thecleavage of EphA4 (antibody A) was obtained. The isotype of antibody Awas determined with monoclonal antibody isotyping kit (Serotec) to beIgG₁ for the heavy chain and κ for the light chain.

(C) Sequence Analysis of Antibody A

The DNA sequence encoding the signal sequence and the variable region ofheavy and light chains of antibody A was amplified by 5′-RACE (5′-rapidamplification of cDNA ends) method. Total RNA was prepared from thehybridoma with RNeasy (QIAGEN), and treated with DNase (QIAGEN, RNasefree DNase set). Double-stranded cDNA was prepared from the total RNAwith cDNA synthesis kit (TAKARA). 5′ Adaptor obtained by annealing ofoligoDNA ad29S (ACATCACTCCGT) (SEQ ID NO. 7) and oligoDNA ad29AS(ACGGAGTGATGTCCGTCGACGTATCTCTGCGTTGATACTTCAGCGTAGCT) (SEQ ID NO. 8) wasadded to the cDNA. The cDNA obtained was amplified with 5′ forwardprimer (5′-PCR4 primer, AGCTACGCTGAAGTATCAACGCAGAG) (SEQ ID NO. 9) and3′ reverse primer (GCCAGTGGATAGACTGATGG (SEQ ID NO. 10) was employed foramplification of the mouse IgG heavy chain, and GATGGATACAGTTGGTGCAGC(SEQ ID NO. 11) was employed for amplification of the mouse Igκ lightchain). The amplified cDNA was inserted into pCR2.1 vector(Invitrogen/LifeTechnologies). The gene sequence of antibody A wasanalyzed with ABI 3130XL. As amino acid sequences coded by the genesequence of antibody A identified by the present analysis, the heavychain signal sequence is the sequence shown in SEQ ID NO. 12, the heavychain variable region is the sequence shown in SEQ ID NO. 13, the lightchain signal sequence is the sequence shown in SEQ ID NO. 14, and thelight chain variable region is the sequence shown in SEQ ID NO. 15. Asnucleotide sequences coding the gene sequence of antibody A, heavy chainsignal sequence is the sequence shown in SEQ ID NO. 16, the heavy chainvariable region is the sequence shown in SEQ ID NO. 17, the light chainsignal sequence is the sequence shown in SEQ ID NO. 18, and the lightchain variable region is the sequence shown in SEQ ID NO. 19.

The full length sequences of the heavy and light chains of antibody Awere obtained with the following steps. Total RNA was prepared from thehybridoma with RNeasy (QIAGEN), and treated with DNase (QIAGEN, RNasefree DNase set). Reverse transcription products were prepared from thetotal RNA with RNA PCR kit (TAKARA). Employing the reverse transcriptionproducts obtained as templates, the gene sequence encoding the heavy andlight chains of antibody A was amplified with PCR with 5′ forward primer(GCGAAGCTTGCCGCCACCATGGCTGTCCTGGTGCTGCTCC (primer ID 7455) (SEQ ID NO.20) was used for amplification of the heavy chain, andGCGAAGCTTGCCGCCACCATGGACATGAGGGTTCCTGCTCACG (primer ID 7453) (SEQ ID NO.21) was used for amplification of the light chain) and 3′ reverse primer(GCGGAATTCATCATTTACCAGGAGAGTGGGAGAGGC (primer ID 7257) (SEQ ID NO. 22)was used for amplification of the heavy chain, andCGCGAATTCACTAACACTCATTCCTGTTGAAGCTCTTGAC (primer ID 7249) (SEQ ID NO.23) was used for amplification of the light chain), and respectivelycloned into pEE6.4 and pEE12.4 vectors (Lonza). The gene sequence wasanalyzed with ABI3130XL. As amino acid sequences coded by the genesequence of antibody A identified by the present analysis, the heavychain constant region is the sequence shown in SEQ ID NO. 24, and thelight chain constant region is the sequence shown in SEQ ID NO. 25.

The CDR of antibody A was determined with the following method. Theamino acid sequence of antibody A was numbered according to the Kabatnumbering system with Abysis software (UCL). Based on this numbering,decision was made according to Kabat definition for CDR identification.The amino acid sequences of CDR of antibody A are shown in Table 1.

TABLE 1 Amino acid sequences of CDR of antibody A Name SequenceHeavy chain CDR1 RYGVH (SEQ ID NO. 26) Heavy chain CDR2VIWRGGSTDYNAAFMS (SEQ ID NO. 27) Heavy chain CDR3ESLFGVYYDYGYYSMDY (SEQ ID NO. 28) Light chain CDR1RASQEISGYLS (SEQ ID NO. 29) Light chain CDR2 AASTLDS (SEQ ID NO. 30)Light chain CDR3 LQYASYPLT (SEQ ID NO. 31)

Reference Example 2: Binding Affinity of Anti-EphA4 Monoclonal AntibodyAgainst Mouse and Human EphA4

The binding affinity of antibody A against mouse and human EphA4 wasdetermined by surface plasmon resonance (SPR method) employing BiacoreT200 (GE Healthcare). First, anti-His antibody (GE Healthcare,28-9950-56) was fixed onto a sensor chip CM5. Fixation was performed byamine coupling method employing N-hydroxysuccinimide (NHS) andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), andethanolamine was employed for blocking (sensor chip and fixationreagents were all from GE Healthcare). This was diluted to 3.5 μg/mLwith the fixation buffer (10 mM sodium acetate, pH 4.5), and fixed onthe sensor chip according to the protocol attached to Biacore T200.Mouse or human EphA4 extracellular region-SEAP-His10 was diluted withthe running buffer HBS-EP (GE Healthcare, BR-1001-88), and the solutionwas sent on to a flow cell for 120 seconds for capture (the capturedamount about 10 RU). Subsequently, antibody A serially diluted to therange of 100, 50, 25, 12.5, 6.3, 3.2, 1.6, and 0 nM with HBS-EP wasadded to the sensor chip for 120 seconds, and the binding reaction curveat the time of addition (binding phase, 120 seconds) and after additionhad completed (dissociation phase, 600 seconds) was sequentiallyobserved. After the completion of each observation, 4 M MgCl₂ (60seconds, Wako Pure Chemical) was added to regenerate the sensor chip.Fitting analysis by 1:1 binding model employing BIA evaluation softwareattached to the system was performed on the binding reaction curveobtained, and the binding affinity (KD=kd/ka) against mouse and humanEphA4 was calculated.

The binding affinity of antibody A against mouse and human EphA4 (KDvalue) was 1.32×10⁻⁹ M and 1.19×10⁻⁹ M, respectively (FIG. 1). Otherbinding parameters against mouse and human EphA4 were almost to the sameextent. Accordingly, it is thought that antibody A has the same extentof binding affinity towards mouse and human EphA4.

Reference Example 3: EphA4 Cleavage Enhancement Activity of Anti-EphA4Monoclonal Antibody in Hippocampus Neurons

For antibody A, evaluation of EphA4 cleavage enhancement activityemploying hippocampus neurons was performed following the steps below.Rat hippocampus neurons seeded in a 96-well dish (Falcon) were treatedwith antibody A (2.0, 6.7, and 20 nM) and γ selectase inhibitory drugCompound E (50 nM, Enzo Life Sciences). Twenty-four hours later, thiswas washed with PBS (Wako Pure Chemical), SDS sample buffer (Laemmlisample buffer (Bio-Rad) and 5% 2-mercaptoethanol (Bio-Rad)) was added torecover the cells, and this was boiled for 5 minutes. SDS-PAGE wasperformed with this sample, western blotting with anti-EphA4 monoclonalantibody (Abnova) was performed, the band strength was quantified, andthe value of EphA4 C-terminal fragment/full length EphA4 was calculated.

Antibody A concentration-dependently enhanced EphA4 cleavage reaction inhippocampus neurons (FIG. 2).

Reference Example 4: Mouse EphA4-Mouse Ligand Binding InhibitoryActivity of Anti-EphA4 Monoclonal Antibody

For antibody A, the evaluation of the inhibitory activity of bindingbetween mouse EphA4 and mouse ligand was performed following the stepsbelow. Anti-alkaline phosphatase antibody (Thermo SCIENTIFIC) was coatedonto the wells of a 96-well plate (Nunc). After incubating at 4° C.overnight, wells were blocked at room temperature for one hour with 1%Block ACE (DS Pharma Biomedical). After washing three times with 0.02%Tween20/PBS (Thermo SCIENTIFIC), mouse EphA4 extracellularregion-SEAP-His protein was added to the wells (final concentration 10nM), and this was incubated at room temperature for one hour. Afterwashing three times, the ligand and antibody A (0, 0.003, 0.01, 0.03,0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, and 3000 nM) were added to thewells. Note that biotinylated mouse Ephrin A1-Fc chimera (R&D Systems,final concentration 6 nM) and biotinylated mouse Ephrin B2-Fc chimera(R&D Systems, final concentration 2.5 nM) were employed as ligands.After incubating at room temperature for one hour and washing threetimes, horseradish peroxidase-labeled streptavidin (GE Healthcare) wasadded, and this was incubated at room temperature for one hour. Afterwashing three times, TMBZ (3,3′,5,5′-tetramethylbenzidine, Sigma)solution was added to the wells, and this was incubated at roomtemperature for 2 minutes. An equal amount of the stop solution (1NH₂SO₄, Wako Pure Chemical) was added to the wells, and absorbance at 450nm was read with a microplate reader (PerkinElmer).

Antibody A concentration-dependently suppressed the binding betweenmouse EphA4 and mouse ligand, and the IC₅₀ values against mouse EphrinA1 and Ephrin B2 binding were about 5.9 nM and 3.1 nM, respectively(FIG. 3). Accordingly, it was shown that antibody A strongly inhibitsthe binding between mouse EphA4 and mouse ligand.

Reference Example 5: Human EphA4-Human Ligand Binding InhibitoryActivity of Anti-EphA4 Monoclonal Antibody

For antibody A, the evaluation of the inhibitory activity of bindingbetween human EphA4 and human ligand was performed following the stepsbelow. Anti-alkaline phosphatase antibody (Thermo SCIENTIFIC) was coatedonto the wells of a 96-well plate (Nunc). After incubating at 4° C.overnight, wells were blocked at room temperature for one hour with 1%Block ACE (DS Pharma Biomedical). After washing three times with 0.05STween 20/PBS (Thermo SCIENTIFIC), human EphA4 extracellularregion-SEAP-His protein was added to the wells (final concentration 10nM), and this was incubated at room temperature for one hour. Afterwashing three times, the ligand and serially diluted antibody A (0,0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, and 3000 nM)were added to the wells. Note that biotinylated human Ephrin A5-Fcchimera (R&D Systems, final concentration 0.7 nM) and biotinylated humanEphrin B3-Fc chimera (R&D Systems, final concentration 2.3 nM) wereemployed as ligands. After incubating at room temperature for one hourand washing three times, horseradish peroxidase-labeled streptavidin (GEHealthcare) was added, and this was incubated at room temperature forone hour. After washing three times, TMBZ(3,3′,5,5′-tetramethylbenzidine, Sigma) solution was added to the wells,and this was incubated at room temperature for 2-5 minutes. An equalamount of the stop solution (1N H₂SO₄, Wako Pure Chemical) was added tothe wells, and absorbance at 450 nm was read with a microplate reader(Molecular Devices or PerkinElmer).

Antibody A concentration-dependently suppressed the binding betweenhuman EphA4 and human ligand, and the IC₅₀ values against human EphrinA5 and Ephrin B3 binding were about 2.8 nM and 1.4 nM, respectively(FIG. 4). Accordingly, it was shown that antibody A also stronglyinhibits the binding between human EphA4 and human ligand.

Reference Example 6: Selectivity of Anti-EphA4 Monoclonal AntibodyAgainst Human Eph Receptor

Following the method for preparing mouse EphA4 extracellularregion-SEAP-His protein described in Reference Example 1, the DNAsequences encoding the signal sequence and the extracellular region ofeach Eph receptor of human (EphA1, EphA2, EphA3, EphA4, EphA5, EphA6,EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, and EphB6) wereamplified by RT-PCR with total RNA derived from tissue, and cloned intopENTRIA vector (Invitrogen/LifeTechnologies) having the DNA sequenceencoding SEAP and histidine tag. Next, the DNA sequences encoding thesignal sequence and extracellular region of each Eph receptor of human,SEAP, and histidine tag were transferred to pcDNA 3.1_rfcB vector by LRreaction of Gateway System (Invitrogen/LifeTechnologies) to constructvectors (referred to as “Eph receptor extracellular region-SEAP-Hisprotein expression vector”) expressing a protein having SEAP and His tagfused to the extracellular region of each Eph receptor of human(referred to as “Eph receptor extracellular region-SEAP-His protein.”)

Next, each Eph receptor extracellular region of human-SEAP-His proteinexpression vector was introduced into Expi293F cells(Gibco/ThermoFisher) with Expi293 expression system(Gibco/ThermoFisher). After 5 days of culture (5% CO2, 37° C., 120 rpm),the culture supernatant was recovered, and this was centrifuged at roomtemperature, at 1500 rpm, and for 5 minutes. The centrifuged supernatantwas filtered with a 0.45 μm filter (Millipore).

For antibody A, the evaluation of the binding activity of human Ephreceptor was performed following the steps below.

Rabbit anti-6-His antibody (Bethyl Laboratories) was coated onto thewells of a 96-well plate (Nunc). After incubating at 4° C. overnight,wells were blocked at room temperature for one hour with 11 Block ACE(DS Pharma Biomedical). After washing three times with 0.05 Tween 20/PBS(Thermo SCIENTIFIC), each Eph receptor extracellular region ofhuman-SEAP-His protein (final concentration 1 nM) was seeded in eachwell, and this was incubated at room temperature for one hour. Afterwashing three times, human IgG solution (100 μg/mL, Mitsubishi PharmaCorporation) and antibody A (10 μg/mL) were added to the wells, and thiswas incubated at room temperature for one hour. Horseradishperoxidase-labeled donkey anti-mouse IgG antibody (JacksonImmunoResearch Laboratories) was added, and this was incubated at roomtemperature for one hour. After washing three times, TMBZ(3,3′,5,5′-tetramethylbenzidine, Sigma) solution was added to the wells,and upon confirmation of a moderate amount of coloring, an equal amountof the stop solution (1N H₂SO₄, Wako Pure Chemical) was added to thewells, and absorbance at 450 nm was read with a microplate reader(PerkinElmer).

Among the human Eph receptor family, antibody A had specific bindingactivity only towards human EphA4 (FIG. 5).

Reference Example 7: Selectivity of Anti-EphA4 Monoclonal AntibodyAgainst Mouse Eph Receptor

Following the method for preparing EphA4 extracellular region-Fc-Hisprotein according to Reference Example 1, the DNA sequences encoding thesignal sequence and the extracellular region of each Eph receptor ofmouse (EphA1, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1,EphB2, EphB3, EphB4, and EphB6) were amplified by RT-PCR with total RNAderived from tissue, and cloned into pENTR1A vector(Invitrogen/LifeTechnologies) having the DNA sequence encoding the Fcregion of human IgG, and histidine tag. Next, the DNA sequences encodingthe signal sequence and extracellular region of each Eph receptor ofmouse, Fc, and histidine tag (EphA1, EphA3, EphA4, EphA5, EphA6, EphA7,EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, and EphB6) were transferredto pcDNA 3.1_rfcB vector by LR reaction of Gateway System(Invitrogen/LifeTechnologies) to construct the extracellular region ofeach Eph receptor of mouse-Fc-His protein expression vector. In theconstruction of the extracellular region of mouse EphA2-Fc-His proteinexpression vector, the DNA sequences encoding the signal sequence andthe extracellular region of mouse EphA2 were amplified by RT-PCR withtotal RNA derived from tissue, and cloned into pcDNA 3.1 vector havingthe DNA sequence encoding Fc and histidine tag to construct a mouseEphA2 extracellular region-Fc-His protein expression vector.

Next, each Eph receptor extracellular region of mouse-Fc-His proteinexpression vector was introduced into Expi293F cells(Gibco/ThermoFisher) with Expi293 expression system(Gibco/ThermoFisher). After 5 days of culture (5% CO2, 37° C., 120 rpm),the culture supernatant was recovered, and this was centrifuged at roomtemperature, at 1500 rpm, and for 5 minutes. The centrifuged supernatantwas filtered with a 0.45 μm filter (Millipore).

For antibody A, the evaluation of the binding activity of mouse Ephreceptor was performed following the steps below.

Rabbit anti-6-His antibody (Bethyl Laboratories) was coated onto thewells of a 96-well plate (Nunc). After incubating at 4° C. overnight,wells were blocked at room temperature for one hour with 1% Block ACE(DS Pharma Biomedical). After washing three times with 0.05? Tween20/PBS (Thermo SCIENTIFIC), each Eph receptor extracellular region ofmouse-Fc-His protein (final concentration 1 nM) was seeded in each well,and this was incubated at room temperature for one hour. After washingthree times, human IgG solution (100 μg/mL, Sigma) and antibody A (10g/mL) were added to the wells, and this was incubated at roomtemperature for one hour. Horseradish peroxidase-labeled donkeyanti-mouse IgG antibody (Jackson ImmunoResearch Laboratories) was added,and this was incubated at room temperature for one hour. After washingthree times, TMBZ (3,3′,5,5′-tetramethylbenzidine, Sigma) solution wasadded to the wells, and upon confirmation of a moderate amount ofcoloring, an equal amount of the stop solution (1N H₂SO₄, Wako PureChemical) was added to the wells, and absorbance at 450 nm was read witha microplate reader (PerkinElmer).

Among the mouse Eph receptor family, antibody A had specific bindingactivity only towards mouse EphA4 (FIG. 6).

Reference Example 8: Reactivity of Anti-EphA4 Monoclonal AntibodyAgainst Mouse, Rat, Monkey, and Human EphA4

The preparation of mouse, rat, monkey, and human EphA4 extracellularregions-Fc-His proteins was performed following the steps below. First,following the method for preparing EphA4 extracellular region-Fc-Hisprotein according to Reference Example 1, monkey EphA4 extracellularregion-Fc-His protein expression vector was constructed. The amino acidsequence of monkey EphA4 utilized in vector construction is shown as SEQID NO. 32, and the extracellular region thereof is shown as SEQ ID NO.33. Various EphA4 extracellular region-Fc-His proteins were preparedemploying the monkey EphA4 extracellular region-Fc-His proteinexpression vector as well as the mouse, rat, and human EphA4extracellular region-Fc-His protein expression vectors described inReference Example 1.

For antibody A, the evaluation of the binding activity with variousEphA4 extracellular regions was performed following the steps below.

Donkey anti-human IgG antibody (Jackson ImmunoResearch Laboratories) wascoated onto the wells of a 96-well plate (Nunc). After incubating at 4°C. overnight, wells were blocked at room temperature for one hour with1% Block ACE (DS Pharma Biomedical). After washing three times with0.05% Tween20/PBS (Thermo SCIENTIFIC), mouse, rat, monkey, and humanEphA4 extracellular regions-Fc-His proteins (final concentration 1 nM)were seeded in the wells, and this was incubated at room temperature forone hour. After washing three times, human IgG solution (100 μg/mL,Mitsubishi Pharma Corporation) and antibody A (0, 0.00013, 0.00064,0.0032, 0.016, 0.08, 0.4, 2, and 10 μg/mL) were added to the wells, andthis was incubated at room temperature for one hour. Horseradishperoxidase-labeled donkey anti-mouse IgG antibody (JacksonImmunoResearch Laboratories) was added, and this was incubated at roomtemperature for one hour. After washing three times, TMBZ(3,3′,5,5′-tetramethylbenzidine, Sigma) solution was added to the wells,and upon confirmation of a moderate amount of coloring, an equal amountof the stop solution (1N H?SO₄, Wako Pure Chemical) was added to thewells, and absorbance at 450 nm was read with a microplate reader(PerkinElmer).

Antibody A had equivalent binding activity in all of mouse, rat, monkey,and human EphA4 (FIG. 7).

Reference Example 9: Reactivity of Anti-EphA4 Monoclonal AntibodyAgainst Human EphA4 Extracellular Region, Ligand Binding Domain,Fibronectin Type III Domain 1, Fibronectin Type III Domain 2

The preparation of proteins having the extracellular region (ECD),ligand binding domain (LBD), fibronectin type III domain 1 (FN1), orfibronectin type III domain 2 (FN2) of human EphA4 fused withmaltose-binding protein (MBP) and histidine tag (hereinafter referred toas “human EphA4 extracellular region-MBP-His protein,” “human EphA4ligand binding domain-MBP-His protein,” “human EphA4 fibronectin typeIII domain 1-MBP-His protein,” and “human EphA4 fibronectin type IIIdomain 2-MBP-His protein”) was performed following the steps below.Initially, pcDNA 3.4-human EphA4 extracellular region, ligand bindingdomain, fibronectin type III domain 1, or fibronectin type III domain2-MBP-His expression vectors were constructed. First, the signalsequence of human EphA4 (SEQ ID NO. 34) or the signal sequence ofpreprotrypsin (SEQ ID NO. 35) and the DNA sequences encoding each domainof human EphA4 were amplified by PCR, and cloned into pcDNA 3.4 vectorhaving the DNA sequence encoding MBP and histidine tag(Invitrogen/LifeTechnologies) to construct human EphA4 extracellularregion-MBP-His protein, human EphA4 ligand binding domain-MBP-Hisprotein, human EphA4 fibronectin type III domain 1-MBP-His protein, andhuman EphA4 fibronectin type III domain 2-MBP-His protein expressionvectors. The amino acid sequence of human EphA4 utilized in vectorconstruction is shown as SEQ ID NO. 5, the extracellular region thereofas SEQ ID NO. 36, the ligand binding domain as SEQ ID NO. 37, thefibronectin type III domain 1 as SEQ ID NO. 38, and the fibronectin typeIII domain 2 as SEQ ID NO. 39. The above expression vectors weretransfected into Expi293F cells (Thermo SCIENTIFIC) with Expi293expression system (Thermo SCIENTIFIC). Four days later, the culturesupernatant was recovered, and passed through a 0.45 μm filter(Millipore). Crude purification was performed with Amylose resin (NEB),and the buffer was substituted to PBS (Wako Pure Chemical) with ZebaSpin Desalting column (Thermo SCIENTIFIC). The monomer fraction wasdifferentially purified with Superdex 200 10/300 (GE Healthcare).

For antibody A, the evaluation of the binding activity with variousdomains in human EphA4 was performed following the steps below.

Rabbit anti-6-His antibody (Bethyl Laboratories) was coated onto thewells of a 96-well plate (Nunc). After incubating at 4° C. overnight,wells were blocked at room temperature for one hour with 1% Block ACE(DS Pharma Biomedical). After washing twice with 0.02% Tween 20/PBS(Nacalai Tesque), human EphA4 extracellular region-MBP-His protein,human EphA4 ligand binding domain-MBP-His protein, human EphA4fibronectin type III domain 1-MBP-His protein, and human EphA4fibronectin type III domain 2-MBP-His protein (final concentration 10nM) were seeded in the wells, and this was incubated at room temperaturefor one hour. After washing three times, antibody A (final concentration10 nM) was added to the wells, and this was incubated at roomtemperature for one hour. Horseradish peroxidase-labeled goat anti-mouseIgG Fcγ fragment antibody (Jackson ImmunoResearch Laboratories) wasadded, and this was incubated at room temperature for one hour. Afterwashing five times, TMB solution (KPL) was added to the wells, and uponconfirmation of a moderate amount of coloring, an equal amount of thestop solution (2 N H₂SO₄, Wako Pure Chemical) was added to the wells.Absorbance at 450 nm and 650 nm were read by a microplate reader(PerkinElmer).

Antibody A had binding activity for human EphA4 extracellular region(ECD) and ligand binding domain (LBD) (FIG. 8). There was no reactionfor fibronectin type III domain 1 (FN1) and fibronectin type III domain2 (FN2). Accordingly, it was found that antibody A specifically binds tothe ligand binding domain of the human EphA4 extracellular region.

Reference Example 10: Effect of Anti-EphA4 Monoclonal Antibody onIncreasing the Number of Spines in the Hippocampus Neuron

Preparation of rat hippocampus neurons was performed as described in theabove Reference Example 1 (B). Rat hippocampus neurons were introducedwith EGFP gene employing Nucleofector (Lonza), mixed with rathippocampus neurons without gene introduction, and seeded in a 24-wellplate (Falcon) containing a cover glass (Matsunami Glass Industries)coated with poly-L-lysine.

The counting of spine employing hippocampus neurons was performedfollowing the steps below. Rat hippocampus neurons introduced with EGFPat culture Day 13 seeded in a 24-well plate (Falcon) containing a coverglass (Matsunami Glass Industries) coated with poly-L-lysine weretreated with control antibody (mouse IgG₁; BioLegend) or antibody A (6.7and 20 nM) for 24 hours. The cover glass was then transferred to 2% PFA(Wako Pure Chemical)/4 sucrose (Wako Pure Chemical)/PBS, and this wasleft standing for 20 minutes to fix the cells. After removing thefixative solution and washing the cells three times with PBS, 0.25%Triton X-100 (Wako Pure Chemical)/PBS was added, and cellpermeabilization was performed for 15 minutes. The solution was removed,the cover glass was transferred to 2% BSA (Sigma)/0.25% TritonX-100/Opti-MEM (GIBCO) and subjected to one hour of blocking, afterwhich anti-GFP antibody (Nacalai Tesque) was allowed to react for 1.5hours. After removing the primary antibody solution and washing threetimes with PBS, the secondary antibody was allowed to react for onehour. After removing the secondary antibody solution and washing threetimes with PBS, Prolong Gold antifade reagent (Molecular probes) wasadded for mounting, and observation was performed with LSM800 (ZEISS).The experiment described above was carried out three times, and for eachexperiment, neurons were extracted from two cover glasses, spines oneach dendrite were counted with image analysis software Imaris™(Bitplane), and the number of spines per 10 μm for each neuron wascalculated.

Antibody A increased the number of spines in the hippocampus neuron(FIG. 9). This result shows that antibody Ahas the activity to stabilizespines in hippocampus neurons.

Reference Example 11: Effect of Anti-EphA4 Monoclonal Antibody toSuppress Tau Phosphorylation In Vivo

The evaluation of the effect to suppress tau phosphorylation in vivoemploying tau transgenic mouse (rTg4510) was performed following thesteps below. Tau transgenic mice (rTg4510) were subcutaneouslyadministered twice a week from 20 to 26 weeks-old with antibody A orcontrol antibody prepares by conventional methods by immunizing a mousewith dinitrophenol (mouse anti-dinitrophenol antibody) at a dose of 100mg/kg (10 mL/kg) each. On 3.5 days after the final administration,anesthesia was done with 2% isoflurane (Intervet) and a mix of threetypes of anesthetic drugs (4.0 mg/kg of Dormicum (Astellas Pharma), 0.3mg/kg of Domitor (Nippon Zenyaku Kogyo), and 5.0 mg/kg of Vetorphale(Meiji Seika Pharma)), perfusion was performed under anesthesia with PBS(Wako Pure Chemical) containing 3 units/mL of heparin (Ajinomoto) and 1%phosphatase inhibitor cocktail (Nacalai Tesque), and the cerebralhemisphere was resected. The cerebral hemisphere collected was fixed at4° C. while shaking overnight in 2% paraformaldehyde (TAAB)/0.1 Mphosphate buffer (Wako Pure Chemical). The cerebral hemisphere wassubstituted with 20% sucrose (Wako Pure Chemical)/0.1 M phosphate buffer(Wako Pure Chemical) and subsequently 25% sucrose/0.1 M phosphate buffer(Wako Pure Chemical), and then embedded in Tissue-Tek O.C.T. Compound(Sakura Finetek Japan)/25% sucrose, and frozen with liquid nitrogen.Slices were created at a thickness of 7 μm with cryostat CM1860 (Leica),adhered on a slide glass (Muto Pure Chemicals), air-dried with cold air,and then placed in a sealed bag and stored at −80° C. The slide glassused for immunostaining was thawed, air-dried with cold air, and thenwashed with PBS (Wako Pure Chemical), immersed in 1% BSA (Sigma)/10normal donkey serum (Jackson ImmunoResearch Laboratories)/0.5% TritonX-100 (Wako Pure Chemical)/PBS solution, and subjected to one hour ofblocking, after which anti-phosphorylated tau antibody AT8 (FujirebioEurope N.V.) was allowed to react overnight at ordinary temperatures.After washing three times with PBS, the secondary antibody was allowedto react for one hour. After washing three times with PBS, Prolong Goldantifade reagent (Molecular probes) was placed on the slice and mounted,and observation was performed with LSM700 (ZEISS). The AT8signal-positive area in the hippocampus CA1 stratum radiatum wasmeasured with image analysis software ImageJ, and the proportion of theAT8-positive signal area against the total area was calculated.

Antibody A decreased the signal of phosphorylated tau in the hippocampusCA1 region (FIG. 10). This result shows that antibody A has the activityto suppress the progression of tau pathology in tau transgenic mouse(rTg4510).

Reference Example 12: Epitope Mapping of EphA4-Ligand Binding Domain(EphA4-LBD) by X-Ray Crystal Structure Analysis

The preparation of antibody A-Fab was performed following the stepsbelow. Antibody A at 101.1 mg was dissolved in 0.1 M sodium phosphatebuffer (pH 7.0) comprising 30 mM L-cysteine and 2 mM EDTA at aconcentration of 15 mg/mL. To this antibody solution, papain (Sigma) wasadded at 1/200 amount to the antibody, and enzymatic digestion wasperformed at 37° C. for 18 hours. The antibody A enzymatic digestionjuice was dialyzed against PBS, and the precipitate was removed bycentrifugation (the precipitate produced was redissolved in PBS andmixed with the centrifugation supernatant). Next, the following stepswere performed with the purpose of removing impurities other thanantibody A-Fab.

1) Purification by Protein a Column

This enzymatic digestion solution was applied to 2 mL of ProSep vA HighCapacity (Millipore) equilibrated with PBS, and the pass-throughfraction and the PBS wash fraction were recovered. 2) Affinitypurification employing anti-human IgG Fcγ antibody

An affinity column having the anti-human IgG Fcγ antibody (JacksonImmunoResearch Laboratories) covalently bound to NHS-Activated Sepharose4FF (GE Healthcare) was prepared according to the manual of thisSepharose. The solution recovered in the above 1) was charged to thisaffinity column, and the pass-through solution and the PBS wash solutionthereof were recovered.

3) Purification by Gel Filtration

The pass-through fraction obtained in the above 2) was concentrated withan ultrafiltration membrane. Superose 12 (GE Healthcare) wasequilibrated with PBS, the concentrated sample was applied and separatedand purified. A part of the separated and purified fraction was analyzedwith SDS-PAGE, and the fraction comprising antibody A-Fab with highpurity was recovered and pooled. The sample purified in this way was setas antibody A-Fab.

EphA4-LBD was prepared in order to prepare a complex of antibody A-Faband the antigen EphA4-LBD (Qin H. et al., J. Biol. Chem., 283:29473-29484 (2008)). EphA4-LBD at 0.68 μmol (200 μM, 3.4 mL) andantibody A-Fab at 0.45 μmol (300 μM, 1.5 mL) were mixed so thatEphA4-LBD will have a molar ratio of about 1.5 folds against antibodyA-Fab. Next, the mixed solution was applied to HILOAD26/60 Superdex 75prep grade (GE Healthcare), and eluted with the buffer forchromatography (25 mM Tris/HCl (pH 7.5), 100 mM NaCl). The fractioncomprising the complex was analyzed with SDS PAGE, the fractions havinghigh purity were gathered and concentrated to about 40.8 mg/mL, and thiswas employed for crystallization.

Crystallization of the complex was performed by sitting drop vapordiffusion method with an auto crystallization device Hydra II Plus OneSystem (Matrix Technologies Corp., Ltd.). MRC-2 (Molecular Dimensions)was used as the plate. The composition of the reservoir solution was 100mM HEPES (pH 7.5), 10% Polyethylene Glycol 8000, and 8% Ethylene Glycol,and this reservoir solution and the above complex solution were mixed sothat the volume ratio was 1:1 to generate crystallization droplets. Thecrystallization plate generated was left standing at 20° C.

Upon crystallization under the above condition, crystals having spacegroup P212121, lattice constants a=71.0 Å, b=84.5 Å, and c=116.1 Å wereobtained. Radiation light X-Ray (1.0 Å) was incidented to the crystalsobtained to obtain diffraction data of 1.79 Å. The diffraction data wasprocessed by HKL2000 (HKL Research Inc.), and phase determination wasperformed by molecular substitution method. The program PHASER (version2.5.0, McCoy A. J. et al., J. Appl. Cryst. 40: 658-674 (2007)) includedin CCP4 Software Suite (Collaborative computational project number 4,[CCP4] version 6.5.0, Acta Cryst. D 67: 235-242 (2011)) was employed forthe molecular substitution method. The crystal structure of EphA4-LBD(PDBID:3CKH) and the crystal structure of the Fab region of IgG(PDBID:2VXT (L-chain) and 1FGN (H-chain)) were employed as the searchmodel of the molecular substitution method. A molecular model wasconstructed with the program COOT (Emsley P. et al., Acta Cryst. D 60:2126-2132n (2004)) so as to fit the electron density obtained from thephase determined, and structure refinement was performed with theprogram REFMAC (Murshudov G. N., Acta Cryst. D 53: 240-255 (1997)).

The complex crystal structure of 2.0 Å resolution was obtained bystructural calculation (R=0.212, Rfree=0.258).

The crystal structure of the antibody A-Fab/EphA4-LBD complex obtainedwas analyzed with the interaction detection tool equipped in thecomputational chemical system MOE 2018.0101 (Chemical Computing GroupInc.), and the amino acid residues on EphA4-LBD which are in directcontact with antibody A-Fab were identified (FIG. 11A). The identifiedamino acid residues are Glu51, Gly52, Ile59, Gln71, Cys73, Asn74, Va175,Met76, Glu77, Thr104, Arg106, Leu111, Pro112, Met115, Arg162, Met164,Cys191, Ala193, and Val195. FIG. 11B shows the surface structure ofEphA4-LBD generated with Maestro (version 11.0, Schrodinger, LLC). As aresult, the present inventors concluded that the region where theseamino acid residues are present is the antibody A-Fab binding region inEphA4-LBD.

Example 1: Preparation of Humanized Antibody of Antibody A Preparationof Humanized Anti-EphA4 Antibody

The variable region of the humanized antibody was designed. Based on thehigh homology against the framework region (FR) of antibody A, among theFRs of human antibody, IGHV3-33*03 (SEQ ID NO. 42) and JH6 (SEQ ID NO.43) for the heavy chain and IGKV1-17*01 (SEQ ID NO. 40) and JK4 (SEQ IDNO. 41) for the light chain were selected as the FRs for the humanizedantibody. The amino acids in the FR that interact with the amino acidsof the CDR were then predicted employing a 3D structure prediction modelof mouse antibody A, these were transplanted together with the CDR ofantibody A having Y32F mutation in the CDR1 of the heavy chain (SEQ IDNO. 44, 27, 28, and 29-31), and HK2-42 (SEQ ID NO. 45) was designed asthe heavy chain variable region of the humanized antibody and L1-8 (SEQID NO. 46) was designed as the light chain variable region of thehumanized antibody. The amino acid sequences of the transplanted CDR areshown in Table 2, and the nucleic acid sequences are shown in Table 3.

The constant region of human IgG₂ (SEQ ID NO. 47) was employed as theheavy chain constant region. Human Igκ (SEQ ID NO. 48) was employed asthe light chain constant region. An expression vector (pcDNA 3.4)comprising the gene sequence encoding the amino acid sequence of thehumanized antibody was transfected into Expi293F cells(Gibco/ThermoFisher) with Expi293 expression system(Gibco/ThermoFisher). As the gene sequence encoding the amino acidsequence of the humanized antibody, the nucleic acid sequence shown inSEQ ID NO. 55 was employed as heavy chain variable region, the nucleicacid sequence shown in SEQ ID NO. 56 was employed as light chainvariable region, the nucleic acid sequence shown in SEQ ID NO. 57 wasemployed as the heavy chain constant region, and the nucleic acidsequence shown in SEQ ID NO. 58 was employed as the light chain constantregion, respectively. The amino acid sequence of the full length of theheavy chain (not including the signal sequence) of the humanizedantibody is the amino acid sequence shown in SEQ ID NO. 59, the aminoacid sequence of the full length of the light chain (not including thesignal sequence) is the amino acid sequence shown in SEQ ID NO. 60. Thenucleic acid sequence encoding the full length of the heavy chain of thehumanized antibody is the nucleic acid sequence shown in SEQ ID NO. 61,and the nucleic acid sequence encoding the full length of the lightchain is the nucleic acid sequence shown in SEQ ID NO. 62. Thesupernatant was recovered, and the humanized antibody of antibody A(antibody B) was purified with MabSelectSuRe (GE Healthcare).

TABLE 2 Amino acid sequences of CDR of antibody B Name SequenceHeavy chain CDR1 RFGVE (SEQ ID NO. 44) Heavy chain CDR2VIWRGGSTDYNAAFMS (SEQ ID NO. 27) Heavy chain CDR3ESLFGVYYDYGYYSMDY (SEQ ID NO. 28) Light chain CDR1RASQEISGYLS (SEQ ID NO. 29) Light chain CDP2 AASTLDS (SEQ ID NO. 30)Light chain CDR3 LQYASYPLT (SEQ ID NO. 31)

TABLE 3 Nucleic acid sequences of CDR of antibody B Name SequenceHeavy chain CDR1 AGATTTGGAGTGCAT (SEQ ID NO. 49) Heavy chain CDR2GTGATCTGGAGGGGAGGATCCACCGACTACAA CGCTGCTTTTATGAGC (SEQ ID NO. 50)Heavy chain CDR3 GAGAGCCTGTTCGGCGTGTACTATGACTACGGCTACTATTCTATGGATTAT (SEQ ID NO. 51) Light chain CDR1CGCGCCTCCCAGGAGATCTCTGGCTACCTGT CC (SEC ID NO. 52) Light chain CDR2GCTGCCTCCACCCTGGACTCT (SEC ID NO. 53) Light chain CDR3CTGCAGTACGCTTCCTATCCACTGACC (SEQ ID NO. 54)

Example 2: Affinity of Humanized Anti-EphA4 Monoclonal Antibody AgainstHuman EphA4

The binding affinity of antibody B obtained in Example 1 against humanEphA4 was determined by surface plasmon resonance (SPR method) employingBiacore T200 (GE Healthcare). First, anti-His antibody (GE Healthcare,28-9950-56) was fixed onto a sensor chip CM5. Fixation was performed byamine coupling method employing N-hydroxysuccinimide (NHS) andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), andethanolamine was employed for blocking (sensor chip and fixation reagentwere all from GE Healthcare). This was diluted to 3.5 μg/mL with thefixation buffer (10 mM sodium acetate, pH 4.5), and fixed on the sensorchip according to the protocol attached to Biacore T200. Human EphA4extracellular region-SEAP-His10 was diluted with the running bufferHBS-EP (GE Healthcare, BR-1001-88), and the solution was sent onto aflow cell for 120 seconds for capture (the captured amount about 10 RU).Subsequently, antibody B serially diluted to the range of 100, 50, 25,12.5, 6.3, 3.2, 1.6, and 0 nM with HBS-EP was added to the sensor chipfor 120 seconds, and the binding reaction curve at the time of addition(binding phase, 120 seconds) and after addition had completed(dissociation phase, 600 seconds) was sequentially observed. After thecompletion of each observation, 4 M MgCl₂ (60 seconds, Wako PureChemical) was added to regenerate the sensor chip. Fitting analysis by1:1 binding model employing BIA evaluation software attached to thesystem was performed on the binding reaction curve obtained, and theaffinity (KD=kd/ka) against human EphA4 was calculated.

The binding affinity of antibody B against human EphA4 (KD value) was1.34×10⁻⁹ M (FIG. 12). It was shown that antibody B shows an almostequivalent affinity as antibody A which is the antibody beforehumanization.

Example 3: EphA4 Cleavage Enhancement Activity of Humanized Anti-EphA4Monoclonal Antibody in Hippocampus Neurons

For antibody B obtained in Example 1, evaluation of EphA4 cleavageenhancement activity employing hippocampus neurons was performedfollowing the steps below.

Rat hippocampus neurons seeded in a 96-well dish (Falcon) were treatedwith antibody B (2.0, 6.7, and 20 nM) and γ selectase inhibitory drugCompound E (50 nM, Enzo Life Sciences). Twenty-four hours later, thiswas washed with PBS (Wako Pure Chemical), SDS sample buffer (Laemmlisample buffer (Bio-Rad) and 5% 2-mercaptoethanol (Bio-Rad)) was added torecover the cells, and this was boiled for 5 minutes. SDS-PAGE wasperformed with this sample, western blotting with anti-EphA4 monoclonalantibody (Abnova) was performed, the band strength was quantified, andthe value of EphA4 C-terminal fragment/full length EphA4 was calculated.

Antibody B concentration-dependently enhanced EphA4 cleavage reaction inhippocampus neurons (FIG. 13)

Example 4: Human EphA4-Human Ligand Binding Inhibitory Activity ofHumanized Anti-EphA4 Monoclonal Antibody

For antibody B obtained in Example 1, the evaluation of the inhibitoryactivity of binding between human EphA4 and human ligand was performedfollowing the steps below. Anti-alkaline phosphatase antibody (ThermoSCIENTIFIC) was coated onto the wells of a 96-well plate (Nunc). Afterincubating at 4° C. overnight, wells were blocked at room temperaturefor one hour with 1 Block ACE (DS Pharma Biomedical). After washingthree times with 0.05% Tween 20/PBS (Thermo SCIENTIFIC), human EphA4extracellular region-SEAP-His protein (final concentration 10 nM) wasseeded in the wells, and this was incubated at room temperature for onehour. After washing three times, the ligand and serially dilutedantibody B (0, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300,1000, and 3000 nM) were added to the wells. Note that biotinylated humanEphrin A5-Fc chimera (R&D Systems, final concentration 0.7 nM) andbiotinylated human Ephrin B3-Fc chimera (R&D Systems, finalconcentration 2.3 nM) were employed as ligands. After incubating at roomtemperature for one hour and washing three times, horseradishperoxidase-labeled streptavidin (GE Healthcare) was added, and this wasincubated at room temperature for one hour. After washing three times,TMBZ (3,3′,5,5′-tetramethylbenzidine, Sigma) solution was added to thewells, and this was incubated at room temperature for 2-5 minutes. Anequal amount of the stop solution (1N H₂SO₄, Wako Pure Chemical) wasadded to the wells, and absorbance at 450 nm was read with a microplatereader (Molecular Devices or PerkinElmer).

Antibody B concentration-dependently suppressed the binding betweenhuman EphA4 and human ligand, and the IC₅₀ values against human EphrinA5 and Ephrin B3 binding were about 4.9 nM and 1.6 nM, respectively.Accordingly, it was found that antibody B strongly inhibits the bindingbetween human EphA4 and human ligand, and shows an almost equivalentinhibitory activity as antibody A which is the antibody beforehumanization (FIG. 14).

Example 5: Mouse EphA4-Mouse Ligand Binding Inhibitory Activity ofHumanized Anti-EphA4 Monoclonal Antibody

For antibody B obtained in Example 1, the evaluation of the inhibitoryactivity of binding between mouse EphA4 and mouse ligand was performedfollowing the steps below. Anti-alkaline phosphatase antibody (ThermoSCIENTIFIC) was coated onto the wells of a 96-well plate (Nunc). Afterincubating at 4° C. overnight, wells were blocked at room temperaturefor one hour with 1 Block ACE (DS Pharma Biomedical). After washingthree times with 0.024 Tween 20/PBS (Thermo SCIENTIFIC), mouse EphA4extracellular region-SEAP-His protein was added to the wells (finalconcentration 10 nM), and this was incubated at room temperature for onehour. After washing three times, the ligand and serially dilutedantibody B (0, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300,1000, and 3000 nM) were added to the wells. Note that biotinylated mouseEphrin A1-Fc chimera (R&D Systems, final concentration 6 nM) andbiotinylated mouse Ephrin B2-Fc chimera (R&D Systems, finalconcentration 2.5 nM) were employed as ligands. After incubating at roomtemperature for one hour and washing three times, horseradishperoxidase-labeled streptavidin (GE Healthcare) was added, and this wasincubated at room temperature for one hour. After washing three times,TMBZ (3,3′,5,5′-tetramethylbenzidine, Sigma) solution was added to thewells, and this was incubated at room temperature for 2 minutes. Anequal amount of the stop solution (1N H₂SO₄, Wako Pure Chemical) wasadded to the wells, and absorbance at 450 nm was read with a microplatereader (Molecular Devices or PerkinElmer).

Antibody B concentration-dependently suppressed the binding betweenmouse EphA4 and mouse ligand, and the IC₅₀ values against mouse EphrinA1 and Ephrin B2 binding were about 8.7 nM and 4.2 nM, respectively.Accordingly, it was found that antibody B strongly inhibits the bindingbetween mouse EphA4 and mouse ligand, and shows an almost equivalentinhibitory activity as antibody A which is the antibody beforehumanization (FIG. 15).

Example 6: Selectivity of Humanized Anti-EphA4 Monoclonal AntibodyAgainst Human Eph Receptor

Similarly to the method for preparing mouse EphA4 extracellularregion-SEAP-His protein described in Reference Example 1, the DNAsequences encoding the signal sequence and the extracellular region ofeach Eph receptor of human (EphA1, EphA2, EphA3, EphA4, EphA5, EphA6,EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, and EphB6) wereamplified by RT-PCR with total RNA derived from tissue, and cloned intopENTRIA vector (Invitrogen/LifeTechnologies) having the DNA sequenceencoding SEAP protein and histidine tag. Next, the DNA sequencesencoding the signal sequence and extracellular region of each Ephreceptor of human, SEAP protein and histidine tag were transferred topcDNA 3.1_rfcB vector by LR reaction of Gateway System(Invitrogen/LifeTechnologies) to construct vectors (referred to as “Ephreceptor extracellular region-SEAP-His protein expression vector”)expressing a protein having SEAP protein and His tag fused to theextracellular region of each Eph receptor of human (referred to as “Ephreceptor extracellular region-SEAP-His protein.”)

Next, each of human Eph receptor extracellular region-SEAP-His proteinexpression vectors was introduced into Expi293F cells(Gibco/ThermoFisher) with Expi293 expression system(Gibco/ThermoFisher). After five days of incubation (5% CO₂, 37° C.),the culture supernatant was recovered, and this was centrifuged at roomtemperature, at 1500 rpm, and for 5 minutes. The centrifuged supernatantwas filtered with a 0.45 μm filter (Millipore).

For antibody B obtained in Example 1, the evaluation of the bindingactivity of human Eph receptor was performed following the steps below.

Rabbit anti-6-His antibody (Bethyl Laboratories) was coated onto thewells of a 96-well plate (Nunc). After incubating at 4° C. overnight,wells were blocked at room temperature for one hour with 1% Block ACE(DS Pharma Biomedical). After washing three times with 0.05% Tween20/PBS (Thermo SCIENTIFIC), each of human Eph receptor extracellularregion-SEAP-His proteins (final concentration 1 nM) was seeded in eachwell, and this was incubated at room temperature for one hour. Afterwashing three times, human IgG solution (100 μg/mL, Mitsubishi PharmaCorporation) and antibody B (10 μg/mL) were added to the wells, and thiswas incubated at room temperature for one hour. Horseradishperoxidase-labeled donkey anti-human IgG antibody (JacksonImmunoResearch Laboratories) was added, and this was incubated at roomtemperature for one hour. After washing three times, TMBZ(3,3′,5,5′-tetramethylbenzidine, Sigma) solution was added to the wells,and upon confirmation of a moderate amount of coloring, an equal amountof the stop solution (1N H₂SO₄, Wako Pure Chemical) was added to thewells, and absorbance at 450 nm was read with a microplate reader(PerkinElmer).

It was found that antibody B, similarly to antibody A which is theantibody before humanization, specifically binds to human EphA4 amongthe human Eph receptor family (FIG. 16).

Example 7: Selectivity of Humanized Anti-EphA4 Monoclonal AntibodyAgainst Mouse Eph Receptor

Following the method for preparing EphA4 extracellular region-Fc-Hisprotein according to Reference Example 1, the DNA sequences encoding thesignal sequence and the extracellular region of each Eph receptor ofmouse (EphA1, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1,EphB2, EphB3, EphB4, and EphB6) were amplified by RT-PCR with total RNAderived from tissue, and cloned into pENTRIA vector(Invitrogen/LifeTechnologies) having the DNA sequence encoding the Fcregion of human IgG₁ and histidine tag. Next, the DNA sequences encodingthe signal sequence and extracellular region of each Eph receptor(EphA1, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2,EphB3, EphB4, and EphB6) of mouse, Fc, and histidine tag weretransferred to pcDNA 3.1 rfcB vector by LR reaction of Gateway System(Invitrogen/LifeTechnologies) to construct each of mouse Eph receptorextracellular region-Fc-His protein expression vectors. In theconstruction of mouse EphA2 extracellular region-Fc-His proteinexpression vector, the DNA sequences encoding the signal sequence andthe extracellular region of mouse EphA2 were amplified by RT-PCR withtotal RNA derived from tissue, and cloned into pcDNA 3.1 vector havingthe DNA sequence encoding Fc and histidine tag to construct a mouseEphA2 extracellular region-Fc-His protein expression vector.

Next, each of mouse Eph receptor extracellular region-Fc-His proteinexpression vectors was introduced into Expi293F cells(Gibco/ThermoFisher) with Expi293 expression system(Gibco/ThermoFisher). After 5 days of culture (5% CO2, 37° C., 120 rpm),the culture supernatant was recovered, and this was centrifuged at roomtemperature, at 1500 rpm, and for 5 minutes. The centrifuged supernatantwas filtered with a 0.45 μm filter (Millipore).

For antibody B, the evaluation of the binding activity of mouse Ephreceptor was performed following the steps below.

Rabbit anti-6-His antibody (Bethyl Laboratories) was coated onto thewells of a 96-well plate (Nunc). After incubating at 4° C. overnight,wells were blocked at room temperature for one hour with 1% Block ACE(DS Pharma Biomedical). After washing three times with 0.05% Tween20/PBS (Thermo SCIENTIFIC), each of mouse Eph receptor extracellularregion-Fc-His proteins (final concentration 1 nM) was seeded in eachwell, and this was incubated at room temperature for one hour. Afterwashing three times, human IgG solution (100 μg/mL, Sigma) and antibodyB (10 μg/mL) were added to the wells, and this was incubated at roomtemperature for one hour. Horseradish peroxidase-labeled goat anti-humanKappa Light Chain antibody (IBL) was added, and this was incubated atroom temperature for one hour. After washing three times, TMBZ(3,3′,5,5′-tetramethylbenzidine, Sigma) solution was added to the wells,and upon confirmation of a moderate amount of coloring, an equal amountof the stop solution (1N H₂SO₄, Wako Pure Chemical) was added to thewells, and absorbance at 450 nm was read with a microplate reader(PerkinElmer).

Among the mouse Eph receptor family, antibody B had specific bindingactivity only towards mouse EphA4 (FIG. 17).

Example 8: Reactivity of Humanized Anti-EphA4 Monoclonal AntibodyAgainst Mouse, Rat, Monkey, and Human EphA4

For antibody B, the evaluation of the binding activity with variousEphA4 was performed following the steps below.

Anti-alkaline phosphatase antibody (Thermo SCIENTIFIC) was coated ontothe wells of a 96-well plate (Nunc). After incubating at 4° C.overnight, wells were blocked at room temperature for one hour with 12Block ACE (DS Pharma Biomedical). After washing three times with 0.05%Tween 20/PBS (Thermo SCIENTIFIC), mouse, rat, monkey, and human EphA4extracellular region-SEAP-His proteins (final concentration 1 nM) wereseeded in the wells, and this was incubated at room temperature for onehour. After washing three times, human IgG solution (100 μg/mL,Mitsubishi Pharma Corporation) and antibody B (0, 0.00013, 0.00064,0.0032, 0.016, 0.08, 0.4, 2, and 10 μg/mL) were added to the wells, andthis was incubated at room temperature for one hour. Horseradishperoxidase-labeled donkey anti-human IgG antibody (JacksonImmunoResearch Laboratories) was added, and this was incubated at roomtemperature for one hour. After washing three times, TMBZ(3,3′,5,5′-tetramethylbenzidine, Sigma) solution was added to the wells,and upon confirmation of a moderate amount of coloring, an equal amountof the stop solution (1N H₂SO₄, Wako Pure Chemical) was added to thewells, and absorbance at 450 nm was read with a microplate reader(PerkinElmer).

Antibody B had equivalent binding activity in all of mouse, rat, monkey,and human EphA4 (FIG. 18).

Example 9: Reactivity of Humanized Anti-EphA4 Monoclonal AntibodyAgainst Human EphA4 Extracellular Region, Ligand Binding Domain,Fibronectin Type III Domain 1, Fibronectin Type III Domain 2

For antibody B obtained in Example 1, the evaluation of the bindingactivity with various domains in human EphA4 was performed following thesteps below.

Rabbit anti-6-His antibody (Bethyl Laboratories) was coated onto thewells of a 96-well plate (Nunc). After incubating at 4° C. overnight,wells were blocked at room temperature for one hour with 14 Block ACE(DS Pharma Biomedical). After washing twice with 0.02S Tween 20/PBS(Nacalai Tesque), human EphA4 extracellular region-MBP-His protein,human EphA4 ligand binding domain-MBP-His protein, human EphA4fibronectin type III domain 1-MBP-His protein, and human EphA4fibronectin type III domain 2-MBP-His protein (final concentration 10nM) were seeded in the wells, and this was incubated at room temperaturefor one hour. After washing three times, antibody B (final concentration10 nM) was added to the wells, and this was incubated at roomtemperature for one hour. Horseradish peroxidase-labeled rabbitanti-human IgG Fcγ fragment antibody (Jackson ImmunoResearchLaboratories) was added, and this was incubated at room temperature forone hour. After washing five times, TMB (KPL) solution was added to thewells, and upon confirmation of a moderate amount of coloring, an equalamount of the stop solution (2 N H₂SO₄, Wako Pure Chemical) was added tothe wells. Absorbance at 450 nm and 650 nm were read by a microplatereader (PerkinElmer).

Antibody B had binding activity for human EphA4 extracellular region(ECD) and ligand binding domain (LBD) (FIG. 19). There was no reactionfor fibronectin type III domain 1 (FN1) and fibronectin type III domain2 (FN2). Accordingly, it was found that antibody B specifically binds tothe ligand binding domain of the human EphA4 extracellular region.

Example 10: Effect of Humanized Anti-EphA4 Monoclonal Antibody onIncreasing the Number of Spines in the Hippocampus Neuron

Preparation of rat hippocampus neurons was performed as described inReference Example 1 (B). Rat hippocampus neurons were introduced withEGFP gene employing Nucleofector (Lonza), and seeded in a 24-well plate(Falcon) containing a cover glass (Matsunami Glass Industries) coatedwith poly-L-lysine.

The counting of spine employing hippocampus neurons was performedfollowing the steps below. Rat hippocampus neurons introduced with EGFPat culture Day 13 seeded in a 24-well plate (Falcon) containing a coverglass (Matsunami Glass Industries) coated with poly-L-lysine weretreated with control antibody (human IgG₂; Sigma) or antibody B (6.7 and20 nM) for 24 hours. The cover glass was then transferred to 2% PFA(Wako Pure Chemical)/4% sucrose (Wako Pure Chemical)/PBS, and this wasleft standing for 20 minutes to fix the cells. After removing thefixative solution and washing the cells three times with PBS, 0.252Triton X-100 (Wako Pure Chemical)/PBS was added, and cellpermeabilization was performed for 15 minutes. The solution was removed,the cover glass was transferred to 2% BSA (Sigma)/0.25% TritonX-100/OPTI-MEM (GIBCO) and subjected to one hour of blocking, afterwhich anti-GFP antibody (Nacalai Tesque) was allowed to react for 1.5hours. After removing the primary antibody solution and washing threetimes with PBS, the secondary antibody was allowed to react for onehour. After removing the secondary antibody solution and washing threetimes with PBS, Prolong Gold antifade reagent (Molecular probes) wasadded for mounting, and observation was performed with LSM800 (ZEISS).The experiment described above was carried out three times, and for eachexperiment, neurons were extracted from two cover glasses, spines oneach dendrite were counted with image analysis software Imaris®(Bitplane), and the number of spines per 10 μm for each neuron wascalculated.

Antibody B increased the number of spines in the hippocampus neuron(FIG. 20). This result show that antibody B has the activity tostabilize spines in hippocampus neurons.

Example 11: Human EphA4 Cleavage Enhancement Activity of HumanizedAnti-EphA4 Monoclonal Antibody

For antibody B obtained in Example 1, the evaluation of cleavageenhancement activity on human EphA4 was performed following the stepsbelow.

Preparation of rat hippocampus neurons was performed as described inReference Example 1 (B). Rat hippocampus neurons were introduced with ahuman EphA4-HA protein expression vector employing Nucleofector (Lonza),and seeded in a 96-well dish (Falcon) coated with poly-L-lysine. Theseeded rat hippocampus neurons were treated with antibody B (6.7, 20,and 67 nM) and f selectase inhibitory drug Compound E (50 nM, Enzo LifeSciences). About twenty-four hours later, this was washed with PBS (WakoPure Chemical), SDS sample buffer (Laemmli sample buffer (Bio-Rad) and5% 2-mercaptoethanol (Bio-Rad)) was added to recover the cells, and thiswas boiled for 5 minutes. SDS-PAGE was performed with this sample,western blotting with rat anti-HA monoclonal antibody (Roche) wasperformed, the band strength was quantified, and the value of EphA4C-terminal fragment/full length EphA4 was calculated.

Antibody B enhanced human EphA4 cleavage reaction in hippocampus neurons(FIG. 21).

Example 12: Involvement of MMP and ADAM Against the Effect of HumanizedAnti-EphA4 Monoclonal Antibody on Increasing the Number of Spines in theHippocampus Neuron

Preparation of rat hippocampus neurons was performed as described inReference Example 1 (B). A part of rat hippocampus neurons wereintroduced with EGFP gene employing Nucleofector (Lonza), and seeded ina 24-well plate (Falcon) containing a cover glass (Matsunami GlassIndustries) coated with poly-L-lysine.

The counting of spine employing hippocampus neurons was performedfollowing the steps below. Rat hippocampus neurons introduced with EGFPat culture Day 13 seeded in a 24-well plate (Falcon) containing a coverglass (Matsunami Glass Industries) coated with poly-L-lysine weretreated with control antibody (human IgG₂; Sigma) or antibody B (20 nM),as well as DMSO (Sigma) or MMP and ADAM inhibitor GM6001 (2.5 μM,MedChemExpress) for 24 hours. The cover glass was then transferred to 2%PFA (Wako Pure Chemical)/4% sucrose (Wako Pure Chemical)/PBS, and thiswas left standing for 20 minutes to fix the cells. After removing thefixative solution and washing the cells three times with PBS, 0.25%Triton X-100 (Wako Pure Chemical)/PBS was added, and cellpermeabilization was performed for 15 minutes. The 0.25% TritonX-100/PBS was removed, the cover glass was transferred to 2% BSA(Sigma)/0.25% Triton X-100/OPTI-MEM (GIBCO) and subjected to one hour ofblocking, after which anti-GFP antibody (Nacalai Tesque) was allowed toreact for 1.5 hours. After removing the primary antibody solution andwashing three times with PBS, the secondary antibody was allowed toreact for one hour. After removing the secondary antibody solution andwashing three times with PBS, Prolong Gold antifade reagent (Molecularprobes) was added for mounting, and observation was performed withLSM800 (ZEISS). The experiment described above was carried out threetimes, and for each experiment, neurons were extracted from two coverglasses, spines on each dendrite were counted with image analysissoftware Imaris™ (Bitplane), and the number of spines per 10 μm for eachneuron was calculated.

The increase in the number of spines in the hippocampus neuron byantibody B was inhibited by simultaneous treatment with GM6001 (FIG.22). This result shows that antibody B has spine stabilization activityvia MMP and ADAM in hippocampus neurons.

Example 13: Effect of Humanized Anti-EphA4 Monoclonal Antibody toSuppress Tau Phosphorylation In Vivo

The evaluation of the effect to suppress tau phosphorylation in vivoemploying tau transgenic mouse (rTg4510) was performed following thesteps below. Tau transgenic mice (rTg4510) were subcutaneouslyadministered twice a week from 20 to 26 weeks-old with antibody B at adose of 100 mg/kg (10 mL/kg). PBS (Wako Pure Chemical) wassubcutaneously administered at 10 mL/kg for the control group. On 3.5days after the final administration, mice were anesthetized with 2-2.5%isoflurane inhalation anesthetic drug (Intervet) and a mix of threetypes of anesthetic drugs (4.0 mg/kg of Dormicum (Astellas Pharma), 0.3mg/kg of Domitor (Nippon Zenyaku Kogyo), and 5.0 mg/kg of Vetorphale(Meiji Seika Pharma)), perfusion was performed under anesthesia with PBS(Wako Pure Chemical) containing 3 units/mL of heparin (Ajinomoto) and 1%phosphatase inhibitor cocktail (Nacalai Tesque), and the cerebralhemisphere of mice was resected. The cerebral hemisphere collected wasfixed at 4° C. while shaking overnight immersed in 2% paraformaldehyde(TAAB)/0.1 M phosphate buffer (Wako Pure Chemical). The cerebralhemisphere was substituted with 10% sucrose (Wako Pure Chemical)/0.1 Mphosphate buffer (Wako Pure Chemical) and subsequently 20% sucrose/0.1 Mphosphate buffer (Wako Pure Chemical), and then embedded in Tissue-TekO.C.T. Compound (Sakura Finetek Japan)/20% sucrose, and frozen employingan aluminum block cooled with liquid nitrogen. Slices of the cerebralhemisphere were created at a thickness of 7 μm with cryostat CM1860(Leica). The slices were adhered on a silane-coated slide glass (MutoPure Chemicals), air-dried with cold air, and then placed in a sealedbag and stored at −80° C. The slide glass used for immunostaining wastaken out from −80° C., air-dried with cold air, and then washed withPBS (Wako Pure Chemical), immersed in 1% BSA (Sigma)/10% normal donkeyserum (Jackson ImmunoResearch Laboratories)/0.5% Triton X-100 (Wako PureChemical)/PBS solution, and subjected to one hour of blocking, afterwhich anti-phosphorylated tau antibody AT8 (Fujirebio Europe N.V.) wasallowed to react overnight at ordinary temperatures. After washing threetimes with PBS, the secondary antibody was allowed to react for onehour. After washing three times with PBS, Prolong Gold antifade reagent(Molecular probes) was placed on the slice and mounted, and observationwas performed with LSM700 (ZEISS). The AT8 signal-positive area in thehippocampus CA1 stratum radiatum was measured with image analysissoftware Metamorph, and the proportion of the AT8-positive signal areaagainst the total area was calculated.

Antibody B decreased the signal of phosphorylated tau in the hippocampusCA1 region (FIG. 23). This result shows that antibody B has the activityto suppress the progression of tau pathology in tau transgenic mouse(rTg4510).

1. An anti-EphA4 antibody, wherein the anti-EphA4 antibody comprisesheavy and light chains, and comprises: (a) a heavy chain CDR1 consistingof the amino acid sequence shown in SEQ ID NO. 44; (b) a heavy chainCDR2 consisting of the amino acid sequence shown in SEQ ID NO. 27; (c) aheavy chain CDR3 consisting of the amino acid sequence shown in SEQ IDNO. 28; (d) a light chain CDR1 consisting of the amino acid sequenceshown in SEQ ID NO. 29; (e) a light chain CDR2 consisting of the aminoacid sequence shown in SEQ ID NO. 30; and (f) a light chain CDR3consisting of the amino acid sequence shown in SEQ ID NO.
 31. 2. Theanti-EphA4 antibody according to claim 1, wherein the anti-EphA4antibody is humanized. 3.-4. (canceled)
 5. The anti-EphA4 antibodyaccording to claim 1, wherein the heavy chain comprises a variableregion consisting of the amino acid sequence shown in SEQ ID NO. 45, andthe light chain comprises a variable region consisting of the amino acidsequence shown in SEQ ID NO.
 46. 6. The anti-EphA4 antibody according toclaim 1, wherein the heavy chain and the light chain comprise constantregions comprising amino acid sequences from a human antibody.
 7. Theanti-EphA4 antibody according to claim 6, wherein the constant region ofthe heavy chain is the constant region of human IgG.
 8. The anti-EphA4antibody according to claim 7, wherein the constant region of human IgGis the constant region of human IgG₂.
 9. The anti-EphA4 antibodyaccording to claim 8, wherein the constant region of human IgG₂comprises the amino acid sequence shown in SEQ ID NO.
 47. 10. Theanti-EphA4 antibody according to claim 6, wherein the constant region ofthe light chain is the constant region of human Igκ.
 11. The anti-EphA4antibody according to claim 10, wherein the constant region of human Igκcomprises the amino acid sequence shown in SEQ ID NO.
 48. 12. Ananti-EphA4 antibody, wherein the anti-EphA4 antibody comprises heavy andlight chains, the heavy chain comprises the amino acid sequence shown inSEQ ID NO. 59, and the light chain comprises the amino acid sequenceshown in SEQ ID NO.
 60. 13.-17. (canceled)
 18. A pharmaceuticalcomposition comprising the anti-EphA4 antibody according to claim 1 andat least one pharmaceutically acceptable carrier.
 19. (canceled)
 20. Ananti-EphA4 antibody, wherein the anti-EphA4 antibody comprises heavy andlight chains, the heavy chain comprises the amino acid sequence shown inSEQ ID NO. 59, the light chain comprises the amino acid sequence shownin SEQ ID NO. 60, and the C-terminal lysine of the heavy chain isdeleted. 21.-24. (canceled)
 25. A pharmaceutical composition comprisingthe anti-EphA4 antibody according to claim 12 and at least onepharmaceutically acceptable carrier.
 26. A method for inhibiting tauphosphorylation in a subject having Alzheimer's disease, comprisingadministering to the subject an effective amount of the anti-EphA4antibody according to claim
 1. 27. A method for inhibiting tauphosphorylation in a subject having Alzheimer's disease, comprisingadministering to the subject an effective amount of the anti-EphA4antibody according to claim 12.